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We investigated how maternal melatonin is regulated in pregnant rats. To examine the involvement of the conceptus (fetus and placenta) in serum melatonin concentrations, the number of conceptuses was experimentally reduced to one on day 7 of pregnancy (1-conceptus group). Maternal circulating nighttime melatonin levels increased toward day 21 of pregnancy and rapidly decreased to the non-pregnancy levels after parturition, whereas the maternal serum nighttime melatonin levels of the 1-conceptus group on day 21 of pregnancy were significantly lower than normal pregnancy bearing dams more than 10 conceptuses. When the fetuses were removed by fetectomy (all fetuses but not the placentae) on day 12 of pregnancy, serum melatonin concentrations were not decreased. To examine the source of circulating maternal melatonin, mRNA expression of N-acetyltransferase (NAT), which is a late limiting enzyme for melatonin synthesis, was examined in the placenta and fetal pineal. NAT was not expressed in the placenta and was negligible in the pineal gland of the fetus compared with the mother's pineal gland. To examine the effect of placental hormones on maternal melatonin production, a conditioned medium, which was made by incubating placenta of day 20 of pregnancy with medium, was injected into the 1-conceptus dams from day 17 to day 20 of pregnancy. Injection of conditioned medium significantly increased serum melatonin concentrations compared with the control values whereas charcoal treatment abolished the stimulatory effect of conditioned medium. In conclusion, maternal circulating melatonin is from the maternal pineal gland and is increased by placental hormones during pregnancy.