An indirect ELISA was developed by the Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise 'G. Caporale' (IZS A&M) for the detection of Brucella antibodies in cow's milk. Specific monoclonal antibody was used against a bovine IgG(1) epitope and complies with European Commission requirements. The test accuracy was evaluated on milk samples from the regions of Abruzzo and Molise in Italy. The negative samples came from 1,250 officially brucellosis-free herds from the Molise region (Italy). The positive samples were taken from three herds in the Abruzzo and Molise regions where animals positive to the official tests were present and Brucella abortus was isolated. Test specificity was 99.8% (with a confidence interval [CI] of 99.6%-99.9%), while sensitivity was 100% (CI 91.2%-100%). The probability of detecting antibodies in positive milk samples was higher than 50% up to a dilution of 1:256 in negative milk. The probability of identifying an infected herd in the dairy cattle population. Under study was 88.6% (CI 73.9%-95.3%).