The effects of melatonin on the in vivo pituitary LH response to LH-releasing hormone (LHRH) were examined in neonatal male and female rats, in 35- to 44-day-old male rats, and in 35- to 44-day-old male animals which had been either pinealectomized or maintained in constant light for at least 3 weeks before use. Animals were given saline or melatonin (1–100 μg/rat) followed within 30 sec by saline or LHRH (10–1,000 ng/rat) at separate subcutaneous sites. Blood was collected following decapitation either without prior injection or 15, 30, 45, or 60 min afterwards. Serum LH concentrations were determined by double antibody radioimmunoassay. In neonatal male and female rats, melatonin (1 μg) significantly (p < 0.01) suppressed by approximately 65% serum LH at 15 min after LHRH. Suppression was maintained for at least 60 min, a finding which indicates that melatonin blocks rather than delays the response to LHRH. By contrast, in normal, pinealectomized, and constant light older male rats, melatonin (100 μg) had no detectable effect on either the magnitude or the time course of LH release by LHRH. These data extend our previous in vitro findings by demonstrating that melatonin is a potent inhibitor of the in vivo pituitary response to LHRH in neonatal rats but not in older animals. Neither pinealectomy nor constant light, both of which are assumed to reduce pineal melatonin production, for at least 3 weeks before use restores neonatal pituitary responsiveness to the pineal indole in the older a