Antibiotic resistance patterns of the major group of bovine mastitis pathogens (Streptococcus agalactiae, other streptococci, Staphylococcus aureus, and Staphylococcus epidermidis) were examined by determining the minimum inhibitory concentration (MIC) of 13 different antibiotics against bacterial isolates from dairy cattle. The bacterial strains were obtained from milk samples from each cow in 21 New York state dairy herd surveys. In 12 herd surveys (high antibiotic-use group), all 365 cows received antibiotic infusions into the udder at the cessation of each lactation cycle. The 324 animals in the other nine herd surveys (low antibiotic-use group) did not routinely receive antibiotics during the nonlactation period. The MICs from the two groups were compared by calculating for each bacterial group the average MIC, the antibiotic concentration necessary to inhibit 90% of the isolates, and the antibiotic concentration necessary to inhibit 50% of the isolates. Increased resistance to all 13 antibiotics was observed with Streptococcus agalactiae isolates from the high antibiotic use herds. However, there was relatively little difference between the two groups in the resistance patterns of the other bacterial species examined. The most important finding of the study was the identification of a multiple beta-lactam resistance phenotype in Streptococcus agalactiae.