An inbred mouse model for the human N-acetylation polymorphism has been used to investigate the biochemical basis for the arylamine N-acetylation polymorphism and the relationship between the cytosolic enzymes arylamine N-acetyltransferase (NAT), arylhydroxamic acid N,O-acyltransferase, and N-hydroxyarylamine O-acetyltransferase. Biochemical studies of partially purified NAT from rapid and slow acetylator mice revealed identical molecular weights of 31,500, activation energies of 21,000 cal/mol, equivalent affinities for acetyl coenzyme A, broad pH optima, the presence of an active site sulfhydryl group, and similar behavior during purification with anion exchange, gel filtration, and hydrophobic interaction chromatography. The enzymes differed in inhibition by hydrogen peroxide and dithiobis(2-nitrobenzoic acid). These observations taken in conjunction with previous investigations indicate that the rapid and slow mouse NAT enzymes are isozymes with minimal structural differences. NATs from rapid and slow acetylator mice were purified more than 10,000-fold by the following sequence of methods: homogenization and fractional centrifugation, protamine sulfate precipitation, and chromatography on DEAE-Trisacryl M, Sephadex G-100, Amethopterin-AH-Sepharose 4B, butyl agarose, and Sephacryl S-200, with a 15-25% recovery. NAT from B6 mice was purified to greater than 95% purity, as judged by silver staining of sodium dodecyl sulfate-polyacrylamide gels. Although only NAT appeared to be subject to a genetic polymorphism as evidenced by N-acetylation activities in liver cytosol, the purified NAT protein possessed arylhydroxamic acid N,O-acyltransferase, N-hydroxyarylamine O-acetyltransferase, and NAT activities. Thus, the cytosolic N-acetyltransferase of mouse liver may catalyze N-, O-, and N,O-acetyltransfer reactions through a common acetylated intermediate of a single protein.