The purpose of this study was to evaluate the role of the opioid system and the estrogen environment in the nocturnal secretion of melatonin in women with secondary amenorrhea (SA). Nocturnal melatonin concentrations in patients with SA were significantly higher than in normal women (p < 0.01 vs. women with normal menstrual cycles). There were significant negative correlations between cumulative melatonin levels (between 8 p.m. and 8 a.m.) and serum estradiol-17β (r = -0.561, p < 0.01) and between peak serum melatonin values and serum estradiol-17β concentrations (r = -0.608, p < 0.01) in SA. Intravenous administration of a conjugated estrogen (Premarin 20 mg) significantly suppressed nocturnal melatonin secretion (p < 0.05), but a continuous intravenous infusion of naloxone (1.6 mg/h from 8 p.m. to 6 a.m.), an opiate antagonist, did not affect nocturnal melatonin secretion in SA. Our findings suggest that elevated nocturnal melatonin secretion may be related to low estrogen levels, but that it is not mediated by the opioid system.