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The antimicrobial effect of β-lactam antibiotics, which had β-lactamase inhibitor activity, on Staphylococcus aureus isolated from mastitis was investigated in this study. Out of 166 isolates, 99 isolates (59.6%) produced β-lactamase, and 98 isolates of 99 were β-lactamase positive in above 12.5 ㎍/㎖ MIC of penicillin. In the providence distribution, β-lactamase production rate of 4 providence, Gangwon, Gyeonggi, Chungcheong, and Jeolla was 100%, 65.7%, 58.8%, and 50.0%, respectively. Antibiotic activities of β-lactam antibiotics against lactamase positive isolates also were investigated. Antimicrobial effects of ampicillin/sulbactam or amoxicillin/clavulanic acid treated group were better than ampicillin or amoxicillin treated group. In antimicrobial effects on intracellular S aureus, there was no difference 1 hour and 4 hour treatment in control, ampicillin, and amoxicillin group, but in 18 hours treatment, ampicillin/sulbactam or amoxicillin/clavulanic acid had a better effect than ampicillin or amoxicillin (p<0.05).