Summary The presence of residues of antimicrobial substances in milk can give rise to problems from the point of view of public health (allergies, digestive disorders, resistance to medicines, etc.), in addition to being the cause of interference in fermentation processes used in the dairy industry. It is therefore necessary to set out control measures to ensure that milk is free of residues, in compliance with the legislation laid down by the European Union. Faced with the possible lack of reliability of the methods for inhibitor detection in ewe milk, the scant number of works available on this species and the drawbacks caused by the presence of inhibiting substances, both for consumers and the technological processes, the need arose to carry out a study on a specific method of sifting or "screening" for inhibitor detection (Eclipse( 100ov), as well as the application of a multi-plate or multi-residue microbiological technique (System Microbiological Multi-plate, SMMP) for the confirmation and timely identification of antimicrobial residues in ewe milk. The Eclipse( method is a microbiological agar diffusion technique, containing spores of Bacillus stearothermophilus var calidolactis C953, with an acid-base indicator. Milk is placed in the microplate and, after incubation, the results are interpreted. If the milk does not have inhibitor residues, development of the micro-organism will take place and a change will appear in the colouring of the acid-base indicator present in the medium, the initial blue colour turning yellow. On the other hand, if inhibitors are present at a suitable concentration to prevent the development of the micro-organism, the colour change will not come about. The "System Microbiological Multi-plate" or multi-residue (SMMP) is a microbiological method that selectively detects the presence of residues of different antimicrobial groups in milk, which is analysed through different Petri dishes with a specific composition, in order to optimise the growth conditions of the micro-organism most sensitive to each group of substances. To this end, B. Stearothermophilus var. calidolactis C953 (beta-lactams), B. subtilis BGA at pH 8,0 (aminoglucosides), M. luteus ATCC 9341 (macrolides), E. coli ATCC 11303 (quinolones), B. cereus ATCC 11778 (tetracyclines) y B. subtilis BGA, a pH 7,0 (sulphonamides) are all used. The presence of antimicrobial residues is manifested by the appearance of a microbial growth inhibition zone. The experiments were performed on a total of 27 antimicrobial substances belonging to the beta-lactam groups (penicillin G, amoxycillin, cloxacillin, oxacillin, cephalexin, cephadroxyl, cephoperazone and cephuroxime), aminoglucosides (gentamycin, kanamycin, neomycin and streptomycin), macrolides (erythromycin, spiramycin and tylosin), quinolones (ciprofloxacin, enrofloxacin, flumequin and norfloxacin), tetracyclines (chlortetracyclin, doxycyclin, oxytetracyclin and tetracyclin) and sulphonamides (sulphadimetoxine, sulphamethazine, sulphanylamide and sulphathiazol). In order to carry out these tests, ewe milk samples were taken from healthy animals, not treated with any medicinal substances, that presented a suitable chemical composition and hygiene-health quality. The parameters evaluated, in both methods, were on the one hand the specificity, either by the study of selectivity, which indicates the presence of "false positive" results, and, in the case of the SMMP, also included a study of crossed specificity to analyse the influence of the antimicrobial substances on the different plates not destined to their detection. On the other hand, the detection limits and their comparison with the Maximum Residue Limits (MRLs) set out in the European legislation were calculated for both methods. As for the Eclipse( "100ov" method, it must be pointed out that no positive ("false positive") results were found in any case. The selectivity relation for the Eclipse(r) "100ov" method throughout the experiment was very high, being 0.99 % for samples without preservative and 0.91 % for those containing acidiol, so indicating the interference of this preservative in the analysis of inhibitors in milk. The antimicrobial detection limits assayed by means of the Eclipse "100ov" technique were close to the MRLs laid down by the EU, these being: 7 (g/kg amoxycillin, 68 (g/kg cloxacillin, 28 (g/kg oxacillin, 5 penicillin "G", 115 (g/kg cephalexin, 110 (g/kg cephoperazone, 170 (g/kg sulphadimetoxine and 250 (g/kg sulphathiazole. However, for the rest of the substances assayed, the detection limits were quite far removed from their respective MRLs. It is therefore necessary to improve the sensitivity of the method, in order to be able to broaden the spectrum of antimicrobial substances to be detected. As for SMMP in ewe milk, the regression equations that relate the concentration and the measurement of the microbial growth inhibition zone present high regression coefficients (0.9719-0.9995) in all cases. This points to a great degree of linearity in the response and the possibility of establishing a diameter or decision level of concentrations close to the established MRLs. The diameters of the inhibition zone considered as decision levels for the different antimicrobial groups were: 20 mm for the beta-lactams, aminoglucosides, macrolides, tetracyclines and sulphonamides, and 22 mm in the quinolones. The SMMP specificity was assessed by selectivity, which was very high at 98-100%, and by crossed specificity. In the study of the latter, significant interference in the dishes given over to detection of beta-lactams by antibiotics of the aminoglucosides group and the macrolides, with special emphasis on tylosin, which presented an inhibition diameter greater than 24 mm for all concentrations. In the remaining dishes, the specificity was good, with slight interference at high concentrations of some of the antibiotics. Overall, it may be concluded that the methods studied are valid, especially for the analysis of beta-lactams and sulphonamides, which is why they could be useful in the sifting analysis, as well as the confirmation and identification of antibiotic residues in ewe milk. Nevertheless, it must be emphasised that it is considered necessary to continue and further the study of detection methods for antimicrobial residues in milk, as well as the development of new methods with greater sensitivity that may provide quality control laboratories and the dairy industry with an effective tool to prevent these residues reaching the consumer, in compliance with current legislation.