The molecular genetic basis of N-acetylation polymorphism has been investigated in inbred mouse models of the human acetylation polymorphism. Two genomic clones, Nat1 and Nat2, were isolated from a C57BL/6J (B6) mouse (rapid acetylator) genomic library. The Nat1 and Nat2 genes both have intronless coding regions of 870 nucleotides and display greater than 47% deduced amino acid similarity with human, rabbit, and chicken N-acetyltransferases. Amplification of Nat1 and Nat2 from A/J (A) mouse (slow acetylator) genomic DNA by the polymerase chain reaction and subsequent sequencing revealed that Nat1 was identical in B6 and A mice, whereas Nat2 contained a single nucleotide change from adenine in B6 to thymine in A mice. This nucleotide substitution changes the deduced amino acid at position 99 from asparagine in B6 to isoleucine in A mice. Hydropathy analysis revealed that this amino acid change alters the hydropathy of the flanking peptide segment in NAT2 from hydrophilic in the B6 mouse to hydrophobic in the A mouse. The amino acid change occurs in a region of the gene where no polymorphism has yet been reported in human or rabbit NAT2 and may represent an important structural domain for N-acetyltransferase activity. Nat1 and Nat2 have the same 5' to 3' orientation in the B6 mouse; the two genes are separated by approximately 9 kilobases, with Nat1 located 5' of Nat2.