The metabolism of methylene chloride (75-09-2) was compared in vitro in liver and lung fractions of male B6C3F1 mice, Fischer 344 rats, Charles River Lake View hamsters, and 4 human accident victims (liver tissue only) to attempt to explain marked species specificity of tumorigenic response in these tissues. The cytochrome P450 and glutathione-S-transferase dependent pathways, respectively, of isolated microsomal and cytosolic fractions were assessed by carbon monoxide formation (cytochrome P450 pathway) and by formaldehyde production (glutathione-S- transferase pathway) after incubation of the tissues with methylene chloride. Cytochrome P450 biotransformation of methylene chloride to carbon monoxide in mouse and hamster lung and liver was significantly more efficient than that of rat or human (liver only). The rate of methylene chloride conversion to carbon monoxide via glutathione-S-transferase in the mouse liver (Vmax 36.4 nmoles/min/mg protein) markedly exceeded that of any other tissue, being 12 times more active than that of the rat (Vmax 2.9 nmoles/min/mg). Neither human nor hamster liver cytosolic fractions showed any detectable methylene chloride metabolism by this pathway, although positive controls confirmed viability of the system. Slight evidence of such metabolism was detected in mouse lung tissue (Vmax 0.15 nmoles/min/mg), but was barely perceived in that of rat or hamster (human lung was not available). In that known carcinogenicity correlates with methylene chloride metabolism by the glutathione pathway in experimental animals, but not the cytochrome P450 pathway, the relative inactivity of that pathway in humans suggests a low risk of methylene chloride-induced carcinogenicity.