To clarify the role the opioid system plays in mediating the stimulation of prolactin release by melatonin, we performed two separate studies between the 6th and 8th days of the follicular phase in normal young women. In the first study, serum prolactin concentrations were determined every 20 min over a 4-hour period following oral administration of melatonin (1 mg given at 13.00 h) in the presence of continuous intravenous infusion of saline or naloxone (1.6 mg/h), an opiate antagonist. In the second study, plasma β-endorphin concentrations were measured over a 3-hour period following oral melatonin administration (1 mg given at 13.00 h). Oral melatonin administration alone induced a significant increase in serum prolactin concentration, which was maximal 3 h after melatonin administration. Prolactin release following melatonin administration was not affected by continuous intravenous infusion of naloxone. No significant change in plasma β-endorphin concentrations was observed following melatonin administration. These findings strongly suggest that the stimulation of prolactin release by melatonin is not mediated by opioids.