The distribution of 125I-melatonin binding sites in the male Syrian hamster brain was recorded at 3 times over a 24 h period. The binding in the hypothalamus, hippocampus, medulla-pons and midbrain of the hamsters varied significantly over the 24 h period with different patterns and phases. No such variations were observed in the parietal cortex. Daily morning (10.00 h) or late afternoon (18.00 h) injections of melatonin for 28 days markedly increased the serum concentrations of melatonin at all times recorded. Serum concentrations of testosterone were significantly lower in animals injected with melatonin in the late afternoon than in the untreated controls; no such decrease was observed in animals injected in the morning despite the continuously elevated levels of circulating melatonin. The daily melatonin injections did not significantly affect 125I-melatonin binding in the hypothalamus, parietal cortex and medulla-pons. In the midbrain, 125I-melatonin binding decreased regardless of the time of injection. In the hippocampus, morning melatonin injections caused a marked decrease in 125I-melatonin binding at all times recorded whereas melatonin injected in the late afternoon led to a decrease in 125I-melatonin binding at 10.00 h only. These results indicate diurnal variations in 125I-melanotin binding sites in discrete brain areas of the golden hamster, persisting despite prolonged duration of elevated levels of circulating melatonin. The differential effects of timed melatonin injections on the hippocampal 125I-melatonin binding sites are positively correlated with the counter-antigonadal response produced by morning melatonin injections.