The major isozymes from affinity column-purified glutathione transferases isolated from Sprague-Dawley rat liver, kidney, and testis cytosol and also from BALB/c mouse and Syrian golden hamster liver cytosol have been resolved by chromatofocusing and tested for their ability to denitrosate and thus detoxicate the DNA-methylating agents and potential carcinogens nitrosocimetidine and 1,3-dimethyl-2-cyano-1-nitrosoguanidine (CyanoDMNG). The isozymes have been kinetically characterized using a battery of substrates permitting, in the rat and mouse cases, subunit composition identification. It has been found that the rat and mouse isozymes belonging to the mu class are uniquely and highly active in the denitrosation of nitrosocimetidine and CyanoDMNG. A specific set of hamster glutathione transferase isozymes were also found to be active in these reactions. We have identified the reaction products produced by the rat liver 3-4 isozyme activity. The glutathione transferase-mediated degradations of 1-methyl-2-nitro-1-nitrosoguanidine and CyanoDMNG generate one molecule of S-nitrosoglutathione per molecule of denitrosated guanidinium compound produced. In the CyanoDMNG incubations essentially all degradation was via denitrosation; nitrite and glutathione disulfide were minor products. In the 1-methyl-2-nitro-1-nitrosoguanidine case nonenzymic degradation of the nitroso compound in the presence of reduced glutathione was evident but little of this decomposition produced S-nitrosoglutathione or 1-methyl-2-nitroguanidine. In the presence of rat transferase 3-4 isozyme, glutathione-dependent 1-methyl-2-nitro-1-nitrosoguanidine degradation was shifted markedly towards denitrosation with the concomitant production of S-nitrosoglutathione.