Agar dilution minimal inhibitory concentrations (MICs) of penicillin, tetracycline, chloramphenicol, and clindamycin were determined using Wilkens-Chalgren agar for 1,266 clinical isolates of anaerobic bacteria. In addition, a reference strain of Bacteroides fragilis was repeatedly tested and demonstrated the precision of the technique. Fifty-six percent of our Bacteroides melaninogenicus strains were resistant (MIC greater than or equal to 4.0 microgram/ml) to penicillin. Resistance to this antibiotic was also seen among other anaerobes, but the results are more in accord with previous reports. Resistance to tetracycline (MIC greater than or equal to 4.0 microgram/ml) was found in 60% of our isolates. Chloramphenicol proved to be the most effective agent in vitro with only 2.0% of strains resistant (MIC less than or equal to 16 microgram/ml). Only 5% of strains were resistant to clindamycin (MIC greater than or equal to 8.0 microgram/ml), and this included 10 isolates of B. fragilis and 4 of B. melaninogenicus. The incidence of resistance of anaerobic bacteria to these frequently used antibiotics is greater than previous reports and indicates the need for reliable susceptibility testing of anaerobic bacteria.