The purpose of this study was to determine and compare the effects of chronic melatonin treatment in vivo on several aspects of prolactin (PRL) cell activity (PRL synthesis, storage, and release) in vitro in male and female Syrian hamsters. Adult male and female hamsters were maintained on long photoperiod and treated with daily late afternoon injections of melatonin (25 micrograms) or vehicle for 11 weeks. Melatonin treatment resulted in an 85% and 65% decrease in serum PRL levels in male and female hamsters, respectively. There was a similar 79% and 64% decrease in PRL release in vitro from pituitaries of male and female hamsters respectively, treated with melatonin in vivo. Total stored PRL was 75% lower in male hamsters and 62% lower in female hamsters receiving melatonin. The synthesis of PRL by pituitaries from melatonin-treated male hamsters was reduced by 65%, whereas in melatonin-treated females it was decreased by 58%. Both nanomolar and micromolar doses of melatonin in vitro caused a modest but significant decrease (14-19%) in the amount of PRL stored in and released from normal male pituitaries without affecting synthesis. The inhibitory effects of melatonin on PRL cell function appear to be more pronounced in male than in female hamsters suggesting a sexually dimorphic response to this pineal hormone. While melatonin's PRL-inhibitory effects appear to be exerted primarily via indirect neuroendocrine mechanisms, a secondary component of its overall regulation of PRL processing may involve direct pituitary mechanisms as well.