Chloroform is carcinogenic in rodents but is not mutagenic or DNA reactive. Chloroform-induced hepatocarcinogenesis in rodents is believed to be secondary to events associated with cytotoxicity and cell proliferation. Understanding the mechanisms of chloroform toxicity may provide insights into the mechanisms of carcinogenicity. The goal of these studies was to characterize the cytotoxicity of chloroform in male B6C3F1 mouse and F-344 rat hepatocytes in vitro. We used an in vitro suspension-culture system that reproduced the exposure of the liver to chloroform and the expression of toxicity in vivo. Simulations of a physiologically based dosimetry model for chloroform indicated that the livers of mice and rats were exposed to chloroform concentrations up to 5 mM for 3 h after hepatotoxic doses of chloroform. Freshly isolated male mouse and rat hepatocytes were exposed to chloroform in sealed flasks and then cultured for 24 h as monolayers. Following a 2- or 3-h exposure in suspension, chloroform induced concentration-dependent cytotoxicity (lactate dehydrogenase release) in culture at concentrations higher than 1 mM. Cytolethality was not increased under reduced oxygen tension, indicating that reductive metabolism does not contribute to chloroform-induced toxicity. A threshold of 1 mM chloroform was also found for glutathione (GSH) depletion, with a 50% depletion at 3.8 mM after 2 h. Addition of dithiothreitol, a reducing agent, did not prevent chloroform-induced toxicity, indicating that oxidation of sulfhydryl groups is not critical for toxicity. The lack of protein sulfhydryl group depletion is consistent with this conclusion. Cotreatment with the cytochrome P450 inhibitor 1-phenylimidazole prevented both cytolethality and GSH depletion, indicating that metabolism is necessary for chloroform-induced toxicity. Both species exhibited similar sensitivity toward chloroform toxicity, indicating that toxicity is not sufficient to explain different susceptibility in heptocarcinogenicity. As chloroform metabolism is saturated in the micromolar range, our results indicate that both metabolism and exposure of the liver cells to high concentrations of chloroform are required for toxicity.