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Previously we have shown that a new nitrosourea, 2-chloroethyl nitrosocarbamoylcystamine (CNCC), undergoes an extensive metabolism in the rat. Two pairs of plasma metabolites have been identified. This suggested our hypothesis that the metabolic pathway involves the reduction of the disulfur bridge followed by the methylation and the oxidation of the thiol groups. The two first intermediates, i.e. the unoxidized metabolites, could not be detected in vivo. Hence, to better understand and to confirm the proposed mechanism of biotransformation of CNCC, its in vitro metabolism has been studied. Incubation of CNCC with a rat liver homogenate or a 10,000g supernatant fraction leads to the formation of four pairs of metabolites. Among them we have identified the two first intermediates not found in vivo and the oxidized metabolites. These findings, together with the kinetics data, suggest that reduction, methylation, and oxidation are very rapid enzymatic reactions. We also show that, for completion of the reaction, the incubation mixture had to contain a cytosolic thioreductase, a microsomal and cytosolic S-methyltransferase, a microsomal oxidase, and an NADPH generating system. The sum of the amounts of metabolites found in the organic extratable material is less than the amount of CNCC metabolized. We conclude that the biotransformation of CNCC proceeds from two fast competitive mechanisms operative on both the disulfur and the nitroso groups.