In studies designed to evaluate the reactivity of the beta-lactam antibiotics in the rabbit kidney, the binding to cortical macromolecules of isotopically labeled cephaloglycin (highly toxic) was compared in vivo and in vitro with that of cephalothin (minimally toxic) and benzylpenicillin (nontoxic). Three hours after administration of equal doses, the amounts of firmly bound antibiotic in whole cortex and in nuclear, mitochondrial, microsomal and cytosolic fractions of cortex were greatest for cephaloglycin (cortical concentration 7% of that measured at 0.5 hr), intermediate for cephalothin (2%) and least for benzylpenicillin (1%); the amounts of firmly bound antibiotic were unrelated to the earlier, peak cortical concentrations. Binding to cortical microsomes in vitro showed a similar pattern (greatest for cephaloglycin, least for benzylpenicillin); in addition, the binding in vitro of cephaloglycin was decreased by the addition of an NADPH-generating system and was not decreased by piperonyl butoxide. These studies provide evidence that the spontaneous reactivity of the beta-lactam ring may be an important determinant of the nephrotoxicity of the cephalosporins and fail to support the existence of a role of the cytochrome P-450 mixed-function oxidases in this reactivity.