Dichloromethane is tumorigenic in lungs and liver of B6C3F1 mice, but is not tumorigenic in rats or hamsters, and its toxicity is associated with glutathione-dependent bioactivation. The objective of the present studies was to investigate the glutathione-dependent bioactivation of [13C]dichloromethane in mouse, rat, and human liver cytosol and the fate of dichloromethane-derived reactive intermediates with 13C NMR. [13C]Formaldehyde hydrate, [13C]S-(hydroxymethyl)glutathione, and [13C]methanol were identified as metabolites of [13C]dichloromethane. [13C]S-(Chloromethyl)glutathione, a putative intermediate in the glutathione-dependent bioactivation of dichloromethane, or derived adducts were not observed. Moreover, no evidence for the formation of S,S'-methylenebis[glutathione] by reaction of glutathione and formaldehyde under physiological conditions was obtained, although methanol was observed as a product. S,S'-Methylenebis[glutathione] was, however, formed by reaction of glutathione and formaldehyde at pH 1. S-(Chloromethyl)-N-acetyl-L-cysteine methyl ester, a surrogate for S-(chloromethyl)glutathione, was prone to hydrolysis. These results corroborate the finding that formaldehyde is a reactive intermediate formed during the glutathione-dependent bioactivation of dichloromethane that may be involved in the observed tumorigenicity of dichloromethane in susceptible species. The results also indicate that S-(chloromethyl)glutathione is an intermediate in the glutathione-dependent bioactivation of dichloromethane and may also play a role in its mutagenicity and carcinogenicity.