Syrian hamster acetylation capacity is catalysed by twoN-acetyltransferase isozymes (NAT1 and NAT2). Hamster NAT2 (polymorphic) displays acetylator-genotype dependent activity resulting in high, intermediate, and low activity levels in homozygous rapid, heterozygous and homozygous slow acetylators, respectively. A λgt l0 size-selected genomic library was constructed fromEcoRl-digested homozygous slow acetylator Bio. 82.73/H-Patscongenic hamster DNA and screened with a hamsterNAT1probe. A 4.2 kb Eco RI insert from a positive clone was subcloned into pUC 18 and the intron-freeNAT2coding region was sequenced. TheNAT2coding regions from genomic templates of other homozygous rapid and slow acetylator congenic and inbred hamster lines were amplified by the polymerase chain reaction, cloned, and sequenced. TwoNAT2alleles were found, one (NAT2*15) from each homozygous rapid acetylator line and one (NAT2*16A) from each homozygous slow acetylator line.NAT2*15 contained an 870 bp open reading frame encoding a 290 amino acid protein.NAT2*16A was similar except for two silent (T36C and A633G) and one nonsense (C727T) substitutions yielding a 242 amino acid open reading frame. TheNAT215 andNAT2*16A alleles were expressed in Escherichia coli JM105 and the recombinant proteins were characterized. Electrophoretic mobilities of theNAT2*15 andNAT2*16A recombinant hamster proteins differed and correlated with the theoretical molecular weights calculated from their respective open reading frames. NAT2 16A exhibited 500-to 1000-fold lower maximum velocities compared to NAT2 15 for N-acetylation of all arylamine and hydrazine substrates tested. NAT2 16A also catalysed the metabolic activation of iV-hydroxyarylamines and N-hydroxyarylamides at rates 33- and 23-fold lower than NAT2 15. Intrinsic clearance (Vmax/Km) calculations suggest that N-acetylation of p-aminobenzoic acid and 2-aminofluorene in Syrian hamsters is catalysed primarily by NAT2 (NAT2 15) in rapid acetylators but by NAT1 (NAT1 9) in slow acetylators. These results provide a molecular basis for rapid and slow acetylator phenotype in the Syrian hamster