N-Acetylation is a major pathway in the metabolism of hydrazine and arylamine drugs, and has been associated with carcinogen bioactivation. Monomorphic hamster liver N-acetyltransferase (NAT1) cDNA was cloned from hamster liver cells by reverse transcriptase-coupled polymerase chain reaction. The determined nucleotide sequence was identical to that reported for NAT1. The NAT1 coding region was subcloned into the pG1 yeast expression vector, but cell extracts provided only transient acetyltransferase activity. In addition, cDNA was subcloned into the expression vectors pFLAG-ATS and pFLAG-MAC, The latter vectors encoded a tac promoter and appended a low-molecular weight (1 kDa) hydrophilic FLAG; marker peptide to the amino terminus of NAT1. Unexpectedly, periplasmic export of FLAGATS-NAT1 by the ompA signal peptide of pFLAG-ATS proved to be detrimental to enzyme activity. High acetyltransferase activity, however, was obtained when the fusion protein was expressed in the cytosol. Enzyme purified to homogeneity by immunoaffinity chromatography exhibited substrate specificity comparable to that of the hamster-derived protein.