Two experiments were designed to determine which tissues accumulate [3H]-melatonin and the metabolic fate of this hormone in the spotted skunk. Tritiated melatonin was injected into the jugular vein of 10 anesthetized skunks 1-3 h before the onset of darkness and allowed to circulate for 22 min before the vasculature was flushed with saline to clear radioactivity from the blood. Selected tissues were removed from five skunks and oxidized in a Packard Biological Oxidizer which yielded 95 +/- 5% recovery of radioactivity. Relatively high amounts of radioactivity were found in the pineal (367 +/- 304 dpm/mg tissue), ovary (69 +/- 38 dpm/mg), pituitary (89 +/- 56 dpm/mg), liver (107 +/- 29 dpm/mg), and kidney (63 +/- 15 dpm/mg). Relatively small amounts of [3H] were found in different brain regions (approximately 6-7 dpm/mg). The uterus, pancreas, and temporalis muscle also accumulated radioactivity (approximately 13 dpm/mg). The lung retained the least amount of radioactivity (4 +/- 1.3 dpm/mg). In the second experiment, hypothalami, pituitaries, and ovaries were removed from the remaining five females. Radioactivity from these tissues was extracted and subjected to thin-layer chromatography. Melatonin accounted for approximately 70% of the radioactivity recovered while 6-hydroxymelatonin and unidentified more polar compounds made up the majority of the melatonin metabolites. These data indicate that tissues other than the hypothalamus are able to accumulate [3H]-melatonin.