Metabolism of chloroform (CHCl3) by a cytochrome P-450-dependent process to a reactive metabolite may be required to elicit hepatic and renal toxicities. Specific inducers or inhibitors of cytochrome P-450 have been employed frequently as tools to demonstrate this relationship between metabolism and toxicity in the liver. The experiments reported herein were designed to identify the relationship between metabolism and toxicity of CHCl3 in the kidney of rabbits, a species in which renal cytochrome P-450 is induced by phenobarbital. Pretreatment with phenobarbital enhanced the toxic response of renal cortical slices to CHCl3 in vitro as indicated by decreased p-aminohippurate and tetraethylammonium accumulation. Phenobarbital pretreatment also potentiated in vitro 14CHCl3 metabolism to 14CO2 and covalently bound radioactivity in rabbit renal cortical slices and microsomes. Addition of L-cysteine significantly reduced covalent binding in renal microsomes from both phenobarbital-treated and control rabbits and was associated with the formation of the radioactive phosgene-cysteine conjugate 2-oxothiazolidine-4-carboxylic acid (OTZ). Formation of OTZ was enhanced in renal microsomes from phenobarbital-pretreated rabbits. Thus, this in vitro model supports the hypothesis that the kidney metabolizes CHCl3 to the nephrotoxic metabolite, phosgene.