Urine was collected from rats during 12 consecutive daily dark periods and assayed for melatonin and norepinephrine; the phase of the vaginal estrous cycle associated with each urine sample was determined from daily vaginal smears. The proestrous phase of the estrous cycle was consistently associated with significant reductions in the excretions of both compounds. The level of melatonin in any urine sample tended to vary as a function of its norepinephrine content; however, the slope of the curve relating these two compounds in metestrous-diestrous samples differed from that for proestrous-estrous specimens. This difference suggests that factors other than the catecholamine (e.g., gonadal hormones) also affect melatonin secretion. Oophorectomy elevated the melatonin concentration of serum but not that of the pineal; this rise was suppressed by the administration of estrogen plus progesterone. The fate of circulating melatonin (as indicated by the proportion of an exogenous dose excreted into the urine) was not affected by the state of the estrous cycle.