A cDNA clone (designated hamAT101) encoding an arylamine acetyltransferase, AT-1, was isolated from a hamster liver lambda gt11 cDNA library using a specific polyclonal antibody raised against AT-1. The cloned cDNA insert consisted of 1181 nucleotides, including an open reading frame of 870 nucleotides encoding 290 amino acid (Mr 33,503). The isolated cDNA displayed high sequence similarity to those of chicken, rabbit, and human acetyltransferases. In Northern blots, the hamAT101 cDNA probe hybridized to an RNA band of 18S in the livers of both slow and rapid acetylator phenotypes. To confirm that hamAT101 cDNA encodes the monomorphic but not the polymorphic protein, the isolated cDNA was expressed in monkey kidney cells (COS-1 cells) using the vector p91023(B). A protein with a molecular weight similar to that of AT-1 was detected upon Western blotting in the 9000 x g supernatant from the transfected cells. The activity toward four different substrates of the 9000 x g supernatant was also examined. In agreement with the results of purified AT-1, the cDNA-expressed protein exhibited a high capacity for N-acetylation of 4-aminoazobenzene and 2-aminofluorene, and O-acetylation of 2-hydroxyamino-6-methyldipyrido [1,2-a:3',2'-d] imidazole, whereas no activity was found for the N-acetylation of p-aminobenzoic acid. These results, in addition to the RNA blot hybridization, indicate that hamAT101 encodes the hamster acetyltransferase AT-1.