In vitro decomposition of ACNU, 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitros ourea hydrochloride, in various conditions was studied with the use of the 14C-labeled compound. Metabolite A, 3,4-dihydro-7-methylpyrimido[4,5-d]pyrimidin-2(1H)-one (an intramolecular cyclized product), was formed spontaneously in the phosphate buffer (pH 7.4) with simultaneous liberation of the alkylating moiety. With rat liver enzyme preparations, formation of three metabolites was observed. Those were metabolite B, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-3-(2-chloroethyl)urea (a denitrosated product), metabolite C, 1-[(4-amino-2-methyl-5-pyrimidinyl) methyl]-5-hydroxy-3-nitroso-2-imidazolidinone (a product via oxidative dechlorination), and metabolite D, 1-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-5-hydroxy-2-imidazolidinone (a denitrosated product of metabolite C). Formation of metabolite B was catalyzed with both cytosolic and microsomal enzymes, not inhibited with SKF-525A, and partly dependent on nicotinamide adenine dinucleotide phosphate (NADPH). These results suggest that at least two enzymatic steps would be involved in the formation of this product. Metabolites C and D were produced by the microsomal preparation, being dependent on O2 and NADPH, inhibited by CO and SKF-525A, and enhanced by phenobarbital pretreatment. When metabolite C was incubated with cytosolic and microsomal preparations, more efficient formation of metabolite D with the former than the latter was observed. From these results, it was assumed that oxidative dechlorination of ACNU to metabolite C would be catalyzed with the microsomal mixed function oxidase, and metabolite D would be produced via denitrosation process of metabolite C.