An acetylator polymorphism has been described in the mouse and the inbred strains C3H/HeJ and A/HeJ constitute rapid and slow acetylators, respectively. The NAT1, NAT2, and NAT3 genes from C3H/HeJ and A/HeJ acetylator inbred mouse strains were amplified using the polymerase chain reaction, cloned into the plasmid vector pUC19, and sequenced. They were then subcloned into the prokaryotic expression vector pKK223-3 and expressed in Escherichia coli strain JM105. The 870-bp nucleotide coding region of NAT1 and NAT3 did not differ between the rapid and slow acetylator mouse strains, or from that of previously published mouse NAT1 and NAT3 sequences. However, NAT2 did differ between the rapid and slow acetylator strains with an A296 T transition which causes a (Asn99-->Ile) substitution in the deduced amino acid sequence. Recombinant NAT1, NAT2, and NAT3 proteins catalyzed N-, O-, and N,O-acetyltransferase activities. NAT3 catalyzed aromatic amine N-acetyltransferase activities at very low rates, which confirms a previous study. Apparent K(m) and Vmax kinetic constants for N-acetylation were 5- to 10-fold lower for recombinant mouse NAT1 than NAT2. Intrinsic clearances for recombinant mouse NAT1- and NAT2-catalyzed N-acetylation of aromatic amine carcinogens were comparable. Both recombinant mouse NAT1 and NAT2 catalyzed the metabolic activation of N-hydroxyarylamine (O-acetylation) and N-hydroxyarylamide (N,O-acetylation) carcinogens. Recombinant mouse NAT3 catalyzed N,O-acetylation at very low rates, while O-acetylation was undetectable. No difference was observed between rapid and slow acetylator recombinant NAT2 proteins to activate aromatic amines by O- or N,O-acetylation, in substrate specificity, expression of immunoreactive protein, electrophoretic mobility, or N-acetyltransferase Michaelis-Menten kinetic constants. However, the slow acetylator recombinant NAT2 protein was over 10-fold less stable than rapid acetylator recombinant NAT2. These studies demonstrate metabolic activation and deactivation by recombinant mouse NAT1, NAT2, and NAT3 proteins and confirm and extend previous studies on the molecular basis for the acetylation polymorphism in the mouse.