A cDNA clone encoding the full coding region of chicken liver arylamine acetyltransferase (EC 2.3.1.5) was isolated from lambda gt10 cDNA library by screening with the 32P-labeled oligonucleotide deduced from the amino acid sequence reported in the preceding paper. The complete nucleotide sequence of the cDNA was determined, from which the amino acid sequence of N-acetyltransferase was deduced. It consisted of 1,302 nucleotides including a 861-nucleotide region coding for 287 amino acids with a molecular weight of 32,914. Two methods were applied to confirm that the cDNA encoded arylamine N-acetyltransferase. First, mRNA was synthesized in vitro by Bluescript and subsequently translated in vitro. The molecular mass of the translation product was 34 kDa consistent with that of the enzyme purified from the chicken liver. The translated protein was immunoprecipitated by a monoclonal antibody to N-acetyltransferase. Second, the cDNA was inserted into an expression vector pcDL1 and introduced into Chinese hamster ovary cells. The supernatant of the homogenate of transfected cells showed a high level of N-acetyltransferase activity with a substrate specificity comparable to that of the liver enzyme. Northern blot analysis revealed three mRNAs corresponding to 4.7, 2.0, and 1.4 kilobases. The levels of N-acetyltransferase mRNAs were relatively high in the liver and distributed in various tissues. Genomic Southern blot analysis indicated the presence of only one gene in the chicken haploid genome.