The effect of high concentrations of milk protein and milk fat on the specificity rates of several antibiotic residue screening tests was evaluated in this study. Milk was sampled from 60 Jersey and 30 Holstein cows at one milking and analyzed for beta-lactam residues using four antibiotic residue screening tests. Cows selected were not treated with an antibiotic for at least 30 d prior to sample collection, and milk was visibly normal. Before milk collection, quarter foremilk was aseptically sampled for mastitis pathogen analysis. Milk subsamples were analyzed for fat and protein contents, and somatic cell counts (SCC). Ten Jersey and four Holstein cows were infected with one or more mastitis pathogens. Concentrations of milk fat (4.78 vs. 3.39%) and protein (3.81 vs. 3.00%) were greater for Jersey cows compared with Holstein cows. Milk SCC averaged 148,000/ml and did not differ by breed. The specificity rates were greater than 0.9 for three of the four screening tests. Across breeds, there was an increased probability of a false-positive outcome for the Penzyme test (Coulter Food Science, Milwaukee, WI)), with increased milk protein content and decreased SCC. Increased milk fat content was associated with an increased probability of false-positive outcomes for the CITE Snap test (IDEXX Laboratories, Inc., Westbrook, ME). High concentrations of milk protein and milk fat can adversely affect antibiotic residue test performance, but the degree of the effect is dependent upon the analytical method of the screening test.