Objective-To determine frequency and antibiotic susceptibility patterns of bacterial pathogens from cows with mastitis treated at a private practice during a 2-year period. Design-Observational study. Animals-Lactating dairy cows from 47 herds of 40 to 600 cows each. Procedure-Bacteria isolated from milk samples were identified as coliforms, Staphylococcus spp, or Streptococcus spp, using selective media. Antibiotic susceptibility testing was performed, using the disk diffusion method with the following antibiotics: gentamicin,amikacin sulfate, penicillin G, penicillin G-novobiocin, ampicillin, cephalothin sodium, ticarcillin, ceftiofur, lincomycin, erythromycin, pirlimycin hydrochloride, sulfonamide, tetracycline, and polymyxin B. Results-Of 354 samples tested, 82 (23.2%) yielded no growth. Of bacteria isolated, 54 (15.3%) were coliforms, 96 (27.1%) were Staphylococcus spp, and 94 (26.6%) were streptococcus spp. Antibiotic susceptibility testing was performed on 62.4% of all samples cultured. For Staphylococcus isolates, cephalothin was the most effective antibiotic in vitro for which a commercially available preparation exists. Penicillin G-novobiocin was the most effective antibiotic in vitro for Streptococcus isolates. Commercial antibiotic preparations approved for intramammary use were not effective in vitro against coliforms that were found to cause mastitis. Clinical Implications-Mastitis caused by coliform organisms does not respond to commercial preparations intended for intramammary use; however, it may respond to parenterally administered antibiotics. Mastitis caused by Staphylococcus spp or Streptococcus spp should be treated first with a cephalothin or penicillin G-novobiocin preparation.