Abstract We have developed an in vitro model for investigation of nephron heterogeneity and cell type-specific patterns of renal injury. To further validate our model and to study biochemical mechanisms of cephalosporin-induced injury, cytotoxicity of three cephalosporins was studied in freshly isolated proximal tubular (PT) and distal tubular (DT) cells from rat kidney. The three cephalosporins [cephaloridine (CPH), cephalexin (CXN), cephalothin (CTN)] were chosen because they exhibit varying degrees of nephrotoxicity in vivo and contain different functional groups. CPH produced greater amounts of lactate dehydrogenase release from PT cells than either CXN or CTN, indicating greater toxicity of CPH, which agrees with in vivo observations. DT cells were not affected by any of the cephalosporins. Thus, the cephem ring is sufficient to produce PT cell injury but the presence of other functional groups modifies toxicity. SKF-525A and α-tocopherol protected PT cells from both CPH and CTN, suggesting involvement of cytochrome P-450 metabolism and oxidative stress. Both PT and DT cells exhibited transport of CPH or CXN and transport of CPH into PT cells was inhibitable by probenecid, consistent with action of a specific carrier. Transport alone, therefore, cannot account for the cell type specificity pattern in vitro. Effects on intracellular glutathione status, malondaldehyde formation, and uncoupler-stimulated respiration were also investigated, and these generally correlated with cell type specificity patterns but not always with degree of cytotoxicity. These results validate further the isolated PT and DT cells as in vitro models to study cell type-specific renal injury and show a role for oxidative stress, cytochrome P-450 bioactivation, and mitochondrial dysfunctions in cephalosporin-induced PT cell injury.