Arylamine N-acetyltransferase (EC 2.3.1.5) activity was examined using skin from Syrian hamster. Two isozymes of arylamine N-acetyltransferase, designated NAT-1 and NAT-2, were detected on anion-exchange high-performance liquid chromatography analysis. Both enzyme activities had indistinguishable molecular masses (30 kDa), but differed significantly in their specificity toward the aromatic amines including serotonin, dopamine, methoxytryptamine, tryptamine, para-phenetidine, para-aminobenzoic acid, and sulphamethazine. Specifically, NAT-2 but not NAT-1 catalyzed acetylation of dopamine to N-acetyldopamine and acetylation of serotonin to form N-acetylserotonin, a direct precursor of melatonin. The two isozymes were also distinguishable based upon their sensitivity toward methotrexate inhibition (50% inhibiting dose for NAT-1 = 380 microM; NAT-2 > 2 mM). The presence of these two activities in the skin raises new questions about the physiologic role of this enzyme in general and in the skin-specific functions in particular.