Temocillin is a recently developed penicillin with a methoxy group in the 6-alpha position. The in vitro activity of temocillin was studied using 932 recent clinical isolates of aerobic and anaerobic Gram-negative bacilli and Gram-positive cocci, and its activity was compared with that of other beta-lactam antibiotics. Temocillin was active against the Enterobacteriaceae, with narrow ranges of MICs and MIC90 values (less than or equal to 8 mg/L) for all isolates except Serratia marcescens and Enterobacter species. Moreover, the compound was active against 46 multiresistant Enterobacteriaceae strains. The drug was also strongly active against Haemophilus influenzae, with beta-lactamase-producing strains being as susceptible as non-beta-lactamase-producing strains. Temocillin showed no useful activity against Pseudomonas aeruginosa, Acinetobacter or Gram-positive cocci, and had only discrete activity against the Bacteroides fragilis group. In general, temocillin displayed the same spectrum of activity against Enterobacteriaceae as third generation cephalosporins, but with a substantially lower intrinsic activity.