A previously unknown melatonin metabolite was isolated by chloroform extraction and reverse phase HPLC from human and rat urine after administration of synthetic melatonin and characterized by mass spectroscopy and proton magnetic resonance spectroscopy to 1-acetyl-1,2,3,3a,8,8a-hexahydro-8a-hydroxy-5-methoxypyrrolo[2,3-b ]indole, a cyclic isomer of 2-hydroxymelatonin. This isolation was based on the fact that our melatonin antibody (a-MT-K1) cross-reacted against this novel metabolite at a level of 0.1% (melatonin 100%). In our HPLC program for indoles the cyclic 2-hydroxymelatonin eluted at 25 min, separately from synthetic indoles, between 6-hydroxymelatonin (19 min) and melatonin (35 min). In [3H] melatonin studies it was found to be present (at 25 min in our HPLC), accounting for 5% of the urinary metabolites of melatonin in the rat. Since beta-glucuronidase-arylsulfatase treatment of rat urine did not liberate the cyclic 2-hydroxymelatonin this would appear to be excreted into urine as the free form.