ArylamineN-acetyltransferase (NAT) polymorphism in humans has been associated with variation in susceptibility to drug toxicity and cancer. In mice, three NAT isoenzymes are encoded byNat1,Nat2andNat3genes. OnlyNat2has been shown previously to be polymorphic, a single nucleotide substitution causing the slow acetylator phenotype in the A/J strain. We sequenced theNatgenes from inbred (CBA and 129/Ola), outbred (PO and TO) and wild-derived inbred (Mus spretusandMus musculus castaneus) mouse strains and report polymorphism in all threeNatgenes ofM. spretusand inNat2andNat3genes ofM. m. castaneus. Enzymatic activity assays using liver homogenates demonstrated thatM. m. castaneusis a ‘fast’ andM. spretusa ‘slow’ acetylator. Western blot analysis indicated that hepatic NAT2 protein is less abundant inM. spretusthanM. m. castaneus. The new allozymes were expressed in a mammalian cell line and NAT enzymatic activity was measured with a series of substrates. NAT1 and NAT2 isoenzymes ofM. m. castaneusexhibited a higher rate of acetylation, compared with those ofM. spretus. Activity of the NAT3 allozymes was hardly detectable, although theNat3gene does appear to be transcribed, since mRNA was detected by RT–PCR in the spleen. Additional polymorphisms, useful forNat-related genetic studies, have been identified between BALB/c, C57Bl/6J, A/J, 129/Ola, CBA, PO, TO,M. m. castaneusandM. spretusstrains in four microsatellite repeats located close to theNatgenes.