The pattern of melatonin production during the estrous cycle of the rat was measured by monitoring urinary 6-sulphatoxymelatonin (aMT.6S) excretion. Adult rats were maintained under a 14L:10D photoperiod and urine was collected at hourly intervals over a 5-day period using an automated collection system; the concentration of aMT.6S was assayed by RIA and hourly outputs were calculated. Each nightly collection of urine was assigned to an estrous cycle stage as determined by the vaginal smear of the preceding morning. Total aMT.6S excretions (mean +/- SEM) during estrous, metestrous, diestrous, and proestrous stages were 493 +/- 49, 539 +/- 44, 562 +/- 40, and 646 +/- 51 pmol/night, respectively (n = 7). The excretion of aMT.6S was significantly higher on the night of proestrus compared to each of the other stages (P < 0.05). To determine whether estrogen was responsible for the increased aMT.6S excretion during proestrus, rats were studied before and after ovariectomy and following implantation with estradiol implants. Total overnight aMT.6S excretion was reduced by 31% in ovariectomized animals relative to the intact state (P < 0.05) and restored to the intact levels by administration of estradiol (P < 0.05). It was concluded that estradiol can modulate melatonin production in adult rats, and that the changing pattern of aMT.6S excretion throughout the estrous cycle may provide a basis for a functional relationship between pineal activity and reproduction in this species.