The binding affinities of imipenem (N-formimidoylthienamycin) to penicillin-binding proteins (PBSs) of Escherichia coli and Pseudomonas aeruginosa were determined by two different methods in which competition with [¹⁴]benzylpenicillin for the binding sites was measured. By both methods imipenem was shown to have very high binding affinities to PBPs-2 and -4 in E. coli and P. aeruginosa, and appreciable affinities to most of their other major PBPs. But higher concentrations of imipenem were required for binding to the PBPs-3 in these bacteria. More direct information about the antibacterial activity of imipenem was obtained by measuring its inhibition of the peptidoglycan-synthetic enzyme activities of E. coli PBPs. The results of enzyme inhibitions were compatible with those obtained in binding experiments. The antibiotic inhibited the transpeptidase activities of PBPs-1A, -1B and -2, and the D-alanine carboxypeptidase activities of PBPs-4 and -5. The antibiotic also seemed to cause strong inhibition of the transglycosylase activity of PBP-1A by some unknown mechanism. It inhibited the transpeptidase activity of PBP-3 only weakly, which is consistent with the findings that it had low binding affinity to PBP-3 and did not inhibit septum formation by the cells.