The toxicity of three cephalosporin antibiotics to rabbit kidney cells in culture was compared to their known nephrotoxic potential in vivo (cephaloridine > cefazolin > cephalothin). While cephalothin is considered to be a relatively nonnephrotoxic cephalosporin when administered to many species including humans and rabbits, in several in vitro systems involving rabbit renal tissue, cephalothin was comparatively more toxic than anticipated based on in vivo data. Cephalothin is extensively des‐acetylated in rabbits to a less microbiologically active metabolite, desacetylcepha‐lothin. When a microsomal S9 fraction from rabbit kidney was added to the in vitro assay in cultured rabbit renal cells, cephalothin was desacetylated and its toxicity to kidney cells was reduced. The addition of S9 in vitro provided a toxicity ranking of the cephalosporins that correlated with their known in vivo nephrotoxic potentials (cephaloridine > cefazolin > cephalothin). The in vitro detoxification of cephalothin by S9 was blocked by the coadministration of the esterase inhibitor, aminocarb. Desacetyl‐cephalothin was relatively nontoxic to rabbit renal tissue in vitro. These results suggest that the desacetylation of cephalothin in vivo represents a previously unrecognized mechanism of detoxification of this cephalosporin antibiotic. Furthermore, this mechanism of detoxification may be applicable to other acetylated cephalosporins.