A primary route of metabolism of dihalomethanes occurs via glutathione (GSH) transferase-catalyzed conjugation. Mammalian theta class GSH transferases and a group of bacterial dichloromethane dehalogenases are able to catalyze the hydrolytic dehalogenation of dihalomethanes via GSH conjugation and subsequent formation of HCHO. Dihalomethanes have been shown to induce revertants in Salmonella typhimurium TA 1535 expressing theta class GSH transferases. Two mammalian theta class GSH transferases (rat GST 5-5 and human GST T1) and the bacterial dehalogenase DM11 were compared in the in vitro conjugation of CH3Cl and using in vitro assays (HCHO formation) and the S. typhimurium mutagenesis assay with the dihalomethanes CH2Cl2, CH2Br2, CH2BrCl, CH2ICl, CH2I2, and CH2ClF. GSTs 5-5 and TI had similar characteristics and exhibited first-order rather than Michaelis-Menten kinetics for HCHO formation over the range of dihalomethane concentrations tested. In contrast, the DM11 enzyme displayed typical hyperbolic Michaelis-Menten kinetics for all of the compounds tested. A similar pattern was observed for the conjugation of CH3Cl The reversion tests with S. typhimurium expressing DM11 or GST 5-5 showed a concentration-dependent increase in revertants for most of the dihalomethanes, and DM11 produced revertants at dihalomethane concentrations lower than GST 5-5. Collectively, the results indicate that rates of conversion of dihalomethanes to HCHO are not correlated with mutagenicity and that GSH conjugates are genotoxic. The results are compared with the conjugation and genotoxicity of haloethanes in the preceding paper in this issue [Wheeler, J. B., Stourman, N. V., Armstrong, R. N., and Guengerich, F. P. (2001) Chem. Res. Toxicol. 14, 1107-1117]. The halide order appears most important in the dihalomethane conjugation reactions catalyzed by GST 5-5 and less so in GST T1 and DM11, probably due to changes in the rate-limiting steps