When ethylene dibromide (EtBr2) was assayed with the Chinese hamster ovary/hypoxanthine-guanine phosphoribosyl transferase (CHO/HGPRT) system coupled with a rat liver metabolic activation system (S9), which contains Ca2+ (Ca, Mg-S9), the cytotoxicity of EtBr2 was greatly increased over that obtained when NADP was omitted from the Ca, Mg-S9 or when EtBr2 was assayed as a direct-acting agent. However, on a molar basis, the mutagenicity of EtBr2 remained unaffected. The omission of Ca2+ from the Ca, Mg-S9 metabolic activation system (Mg-S9), with either the addition or omission of NADP, caused approximately a 2-fold decrease in the mutagenicity of EtBr2 when compared to the results obtained by using the Ca, Mg-S9 system. The cytotoxicity of EtBr2 was further increased when a purified microsomal fraction, prepared from the S9 fraction, was used in the presence of Ca2+. In the absence of this calcium ion, this metabolic activation system was extremely cytotoxic to Chinese hamster ovary cells even without the presence of a mutagen or promutagen. The cytotoxicity of EtBr2 in the following assay systems decreased in this order: Ca, Mg-microsomes greater than Ca, Mg-S9 greater than S9 greater than direct-acting agent greater than or equal to Ca, Mg-S9 without NADP greater than or equal to Mg-S9 without NADP. Cytotoxicity appears to be NADP-dependent on the presence of NADP in the S9 system, the mutant yield (number of mutants that could be induced) was higher in its absence. Addition of reduced glutathione to Mg-S9 without NADP increased the mutagenicity of EtBr2 to values that did not exceed those obtained when EtBr2 was tested as a direct-acting agent. On a molar basis, ethyl methanesulfonate (EMS) is less cytotoxic but equally as mutagenic as EtBr2. However the mutant yield of EMS was higher than that of EtBr. Inclusion of Ca, Mg-S9 in the assay system had no effect on the biological activities of EMS.