Human arylamine N-acetyltransferases NAT1 and NAT2 are highly polymorphic genes that modify individual susceptibility to cancers caused by exposure to arylamine procarcinogens. Strong similarities exist between rat Nats and human NATs, and rat Nat2 polymorphisms result in slow acetylator phenotype. Recently, a third rat Nat, rNat3*1, was reported. Although in vivo toxicological and carcinogenic studies are often conducted in rats, relatively little is known about Nat sequences among available inbred rat strains. We report here that rNat1 and rNat2 open reading frames (ORFs) in 12 inbred rat strains (ACI, BN, BUF, CDF, COP, DA, LEW, LOU/M, MW, PVG, SHR, WF) corresponded to reference rNat1*13 and rNat2*20. While 10 of the 12 strains had reference rNat3*1 ORFs, strains ACI and COP had a variant rNat3*2 ORF characterized by a G619>T transversion (A207S). The rNat3*2 single nucleotide polymorphism reduced Nat3 protein levels and N- and O-acetyltransferase activity when recombinantly expressed in bacteria. Recombinant expression of rNat3 1 and rNat3 2 in COS-1 cells yielded equivalent protein levels but undetectable catalytic activities. Relative tissue expressions of rNat1, rNat2, and rNat3 mRNAs were assessed in liver and 12 extrahepatic tissues (lung, spleen, kidney, heart, esophagus, stomach, urinary bladder, prostate, colon, duodenum, jejunum, ileum) from male F344 rats exsanguinated prior to sacrifice. Semiquantitative RT-PCR experiments demonstrated that the relative expression of the rNat transcripts in liver and 12 extrahepatic tissues was rNat1 > rNat2, while rNat3 transcripts were not detected. This study concludes that rNat1 and rNat2 are primarily responsible for acetylation phenotype in rats.