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The mercapturic acid pathway is a metabolic route for the detoxication of xenobiotics, processing drugs and toxicants to mercapturic acids (N-acetylcysteine conjugates). An N-acetyltransferase enzyme, NAT8, catalyzes the transfer of an acetyl group from acetyl-CoA to the cysteine amino group, producing a mercapturic acid, which is excreted in the urine. Human NAT8 was recently cloned and expressed but little is known concerning its activity and specificity with regard to substrates of toxicological importance. I expressed human NAT8 in HEK 293T cells and used colorimetric assays with DTNB and HPLC analysis (UV or MS detection) to measure the activity of NAT8 for various cysteine substrates. All of the S-aryl-substituted (benzyl, 4-nitrobenzyl, and menaphthyl) cysteine conjugates tested are substrates for NAT8-catalyzed acetylation, with the exception of S-trityl-cysteine. This information provides insight into the final step in the glutathione-dependent biotransformation of toxicologically significant xenobiotics, such as polycyclic aromatic hydrocarbons.