The in vitro activity of cefotetan, a new cephamycin, was compared with the activities of cefoxitin, cefuroxime, moxalactam, ceftazidime, and piperacillin against 273 recent clinical isolates. The minimum inhibitory concentrations of cefotetan for 90% of Enterobacteriaceae, Haemophilus influenzae, and Neisseria gonorrhoeae were between 0.12 and 2 micrograms/ml; for 90% of Staphylococcus aureus, the minimum inhibitory concentration was 8 micrograms/ml, and for 90% of Bacteroides fragilis, Pseudomonas aeruginosa, and Lancefield group D streptococci it was 128 micrograms/ml or more. The activity of cefotetan against Enterobacteriaceae is comparable to the activities of ceftazidime and moxalactam and four to eight times greater than the activities of cefoxitin and cefuroxime. Cefotetan was approximately one-half as active as cefoxitin and cefuroxime against S. aureus. Although cefotetan was 87% bound to serum protein, serum had little effect on the in vitro antimicrobial activity of this agent.