Thienamycin and clavulanic acid are new β-lactam derivatives with structures markedly different from those of penicillins or cephalosporins. Both derivatives had the same general mode of action as typical β-lactam antibiotics since they bound to precisely the same proteins as [14C]benzylpenicillin. Thienamycin showed high affinity for penicillin-binding proteins 1, 2, 4, 5, and 6 and a lower affinity for protein 3. Protein 2 had the highest affinity for thienamycin, and concentrations from the minimal morphological change concentration (0.1 μg/ml) up to about 0.6 μg/ml resulted in the conversion of Escherichia coli KN126 into large osmotically stable round cells. Above a concentration of 0.6 μg/ml, rapid cell lysis occurred with the release of the cell contents as spheroplasts. Clavulanic acid showed good affinity for penicillin-binding protein 2, moderate affinity for proteins 1, 4, 5, and 6, and low affinity for protein 3. Protein 2 had the highest affinity for clavulanic acid, and concentrations from the minimal morphological change concentration (30 μg/ml) up to about 50 μg/ml produced a mixture of slightly elongated, swollen, bulging, and lemon-shaped cells. Above a concentration of 50 μg/ml, rapid lysis occurred with production of spheroplasts. The properties of thienamycin and clavulanic acid were compared with those of the penicillins, cephalosporins, and amidinopenicillanic acids.