Temocillin, a 6-alpha-methoxy penicillin, inhibited 90% of strains of Escherichia coli, Klebsiella pneumoniae, Citrobacter, Proteus, Providencia, Salmonella, and Shigella at a concentration of less than or equal to 16 micrograms/ml. Haemophilus influenzae and Neisseria gonorrhoea were inhibited by less than or equal to 1 microgram/ml. Changing the medium or pH of the cultures did not alter the minimal inhibitory concentrations, which were similar in broth and human serum, as were the minimal bactericidal concentrations. An increase in inoculum size from 10(5) to 10(7) colony-forming units increased concentration required for inhibition. Temocillin inhibited strains resistant to ampicillin, ticarcillin, cefazolin, cefamandole, and cefoxitin. Most Pseudomonas aeruginosa strains and other Pseudomonas spp. and Acinetobacter spp. were resistant, as were gram-positive organisms. Temocillin was not hydrolyzed by the common plasmid and chromosomal beta-lactamases but inhibited them. The resistance of certain gram-negative bacilli to temocillin seemed to be a result of failure of the molecule to enter through the cell wall, since combination of temocillin with EDTA made Pseudomonas, Acinetobacter, and Enterobacter strains susceptible to low concentrations of the compound.