1,2-Dibromo-[1,2-14C]ethane [carcinogen] was bound irreversibly to DNA when glutathione S-transferase or rat liver cytosolic components were added to incubations of calf thymus DNA and glutathione at 37.degree. C. There was no DNA binding of 1,2-dibromoethane when glutathione was absent or in incubations of DNA with microsomal proteins with or without NADPH, thus supporting the proposal that the major route of DNA binding by 1,2-dibromoethane occurs via conjugation to glutathione. In vitro binding of 1,2-dibromoethane occurred most effectively when the YaYc (or B) isozyme of glutathione S-transferase was included in incubations of DNA with 1,2-dibromoethane and glutathione. Other dihaloalkanes were incubated with DNA in the presence of glutathione S-transferase and [35S]glutathione. Only 1,2-dibromo-3-chloropropane and tris-(2,3-dibromopropyl)phosphate led to significant DNA binding of [35S]glutathione. 1,2-Dibromo-3-chloro-[1,3-14C]propane was bound to DNA when glutathione and glutathione S-transferase were present. Even higher 1,2-dibromo-3-chloropropane binding to DNA occurred when cytosol or microsomes were included in incubations without glutathione. When glutathione was added to incubations containing cytosol and 1,2-dibromo-3-chloropropane, total DNA binding was decreased. The actual amount of DNA binding by dihaloethanes in vivo may be the result of a complicated balance among the opposing roles of glutathione conjugation in detoxicating and activating processes.