The in vitro activity of N-formimidoyl thienamycin was determined against 800 gram-positive and gram-negative aerobic and anaerobic bacteria and compared with the activity of cefoxitin, cefazolin, cefamandole, cefotaxime, moxalactam, ampicillin, cefoperazone, and gentamicin. N-Formimidoyl thienamycin inhibited the majority of organisms at concentrations below 1 microgram/ml. It inhibited methicillin-resistant Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus faecalis. It inhibited beta-lactamase-producing Haemophilus influenzae and Neisseria gonorrhoeae. Unlike other new beta-lactams, it inhibited Listeria. Escherichia coli, Klebsiella pneumoniae, enterobacters, Serratia, indole-positive Proteus, Acinetobacter, Pseudomonas aeruginosa, and Bacteroides resistant to other agents were inhibited. There was minimal effect of inoculum size and aerobic versus anaerobic conditions, and serum had no effect on activity. Most minimal bactericidal concentrations were two- or fourfold greater than the minimal inhibitory concentration. N-Formimidoyl thienamycin showed partial synergy with aminoglycosides against S. aureus, S. faecalis, and many Pseudomonas and Enterobacteriaceae. It was not hydrolyzed by plasmid-mediated and chromosomal beta-lactamases.