The effects of daily afternoon melatonin injections on the accumulation of monoamines were studied in extracts of median eminence, and of caudate nucleus, of intact and ovariectomized Syrian hamsters which were administered pargyline 2 h prior to sacrifice. Although no significant effect of melatonin administration on the serotonin (5HT) accumulation after pargyline could be detected, significantly increased amounts of 5HT and of the 5HT metabolite, 5-hydroxyindole acetic acid, were detected in median eminence and in caudate nucleus of melatonin-injected hamsters not treated with pargyline. In both median eminence and in posterior pituitary, dopamine (DA) concentrations were significantly reduced by melatonin administration. In the median eminence of intact hamsters, the accumulation of DA after pargyline was reduced to 22% of controls by melatonin injections; in ovariectomized hamsters, the accumulation of DA was reduced to 9% of controls by melatonin injections. The accumulation of norepinephrine after pargyline was significantly reduced by melatonin administration only in ovariectomized hamsters. No significant inhibitory effects of melatonin injections could be detected on DA accumulation in caudate nucleus. These data suggest that melatonin injections result in substantial inhibition of daytime DA synthesis in median eminence independently of its effects on gonadal steroids. Paradoxically, melatonin-induced inhibition of median eminence DA activity occurred concomitantly with suppression of pituitary and plasma prolactin (PRL). We conclude that daily afternoon melatonin injections inhibit PRL secretion and interfere with cycles of LH in spite of decreased DA activity in the median eminence.