The fate of 3H-melatonin after its intracerebroventricular administration was studied both in different brain regions and in subcellular fractions. The rate of disappearance of 3H-melatonin from the brain was found to be multiphasic. Forty-eight h after a 3H-melatonin injection, radioactivity was still present in the brain. Nonlinear regression analysis of the data confirmed a very rapid half-life component and (t1/2 = 3.04 min) a slower one (t1/2 = 36 min). We also found a much slower component (t1/2 = 24 h), however. Considerable metabolism of melatonin was detected since only 36.5% of administered radioactivity remained as melatonin at 45 min. The subcellular distribution of the radioactivity present in the brain at all times studied showed that a major proportion of the radioactivity remained in the cytosol and respectively decreasing proportions in the 900g pellet, mitochondrial pellet, and the microsomes. The radioactivity remaining in the cytosol at 45 min was found to coelute with a macromolecule that was resolved by gel filtration and could be displaced by previous melatonin administration. Purified nuclei retained 0.71% of the radioactivity at 45 min; of this total, 73% was KCl extractable. Our data suggest the presence of a binding site in the cytosol and in the nucleus. The presence of 3H-melatonin up to 48 h after its administration may account for melatonin's long-term effects on brain function.