Dichloromethane dehalogenase from Methylophilus sp. DM11 is a glutathione S-transferase homolog that is specifically active with dihalomethane substrates. This bacterial enzyme and rat liver glutathione S-transferases were purified to investigate their relative reactivity with CH2Cl2 and related substrates. Rat liver α class glutathione transferases were inactive and μ class enzymes showed low activity (7-23nmol/min/mg of protein) with CH2Cl2. θ class glutathione transferase 5-5 from rat liver and Methylophilus sp. Dichloromethane dehalogenase showed specific activities of≥1μmol/min/mg of protein. Apparen Kcat/Km were determined to be 3.3×104 and 6.0×104L M-1 S-1 for the two enzymes, respectively. Dideutereo-dichloromethane was processed to dideutereo-formaldehyde, consistent with a nucleophilic halide displacement mechanism. The possibility of a GSCH2X reaction intermediate (GS, glutathione; X, halide) was probed using CH2ClF to generate a more stable halomethylglutathione species (GSCH2F). The reaction of CH2ClF with dichloromethane dehalogenase produced a kinetically identifiable intermediate that decomposed to formaldehyde at a similar rate to synthetic HOCH2CH2SCH2F. 19F-NMR revealed the transient formation of an intermediate identified as GSCH2F by its chemical shift, its triplet resonance, and H-F coupling constant consistent with a fluoromethylthioether. Its decomposition was matched by a stoichiometric formation of fluoride. These studies indicated that the bacterial dichloromethane dehalogenase directs a nucleophilic attack of glutathione on CH2Cl2 to produce a halomethylthioether intermediate. This focuses attention on the mechanism used by θ class glutathione transferases to generate a halomethylthioether from relatively unreactive dihalomethanes.