Biotransformation of [3H]serotonin by cultured hamster skin to 3H-metabolites corresponding to N-acetylserotonin (NAS), melatonin, and 5-methoxytryptamine (5MT) was demonstrated. This process was time-dependent, with the highest production of radioactive NAS and melatonin metabolites after 3 and 5 h of incubation followed by a decrease in the rate of metabolite release into the media. Conversely, the formation of radioactive metabolite corresponding to 5-MT increased gradually during skin culture, reaching the highest level after 24 h of incubation. The production of 3H-metabolites, corresponding to NAS, melatonin, and 5-MT, was stimulated by forskolin with a maximum effect of forskolin at 10 μ concentration. The gas chromatographic/mass spectroscopy analysis of the fraction eluting at the retention time of NAS standard material showed that it contained NAS, further confirming production and release of NAS into the media by hamster skin. Therefore, we conclude that mammalian skin can acetylate serotonin to NAS and postulate that the NAS is further metabolized by the skin to form melatonin which is subsequently transformed to 5-MT.