A standardized tablet diffusion test and a reference agar dilution test was evaluated for susceptibility testing of anaerobic bacteria to beta-lactam antibiotics. 74 freshly isolated anaerobic bacteria and three control strains (Cl. perfringens ATCC 13124 B. fragilis ATCC 25285, B. thetaiotaomicron ATCC 29741) were tested. The in vitro activities of 7 beta-lactam antibiotics were compared with metronidazole and clindamycin. Most active were metronidazole and clindamycin. Cefoxitin had the best activity of the beta-lactam antibiotics, whereas piperacillin and carbenicillin had good activities. High resistance rates were found for penicillin, ampicillin, cefuroxime and cefotaxime. MIC on control strains fell well within range set by the National Committee for Clinical Laboratory Standards (NCCLS). Correlation between MIC and inhibition zone diameters was generally good. Tablet diffusion can be used to divide anaerobic bacteria into three susceptibility categories. In addition all bacterial strains were tested for production of beta-lactamase by a nitrocefin tube test. Beta-lactamase production by the nitrocefin test indicated reduced sensitivity to beta-lactam antibiotics.