The expression of mt1 receptor protein in the rat uterus was investigated using an anti-mt1 polyclonal antibody against the rat mt1 receptor. A melatonin receptor protein of 37 kDa was detectable by Western blotting in the rat uterine membrane preparations. Autoradiography with the melatonin ligand, 2-[125I]iodomelatonin, was used to localize melatonin receptors in the uterus of the estrous rats and to study the changes of melatonin receptors in pregnancy. Melatonin receptors were found to be localized in the estrous rat uterine antimesometrial stroma. As decidualization of the uterine stroma progressed during pregnancy, the melatonin binding sites were progressively reduced and became confined to the antimesometrial non-decidualized outer stroma. 2-[125I]Iodomelatonin binding sites were not seen in the mesometrial stromal cells during pregnancy. The role of ovarian hormones in the regulation of uterine melatonin receptors was examined by studying the binding at various phases of the estrous cycle, after ovariectomy with and without follow-on treatment of estradiol (E2), progesterone (P4) or both. 2-[125I]Iodomelatonin binding in the rat uterus fluctuated during the estrous cycle, being lowest during metestrus. Ovariectomy caused an almost 70% reduction of 2-[125I]iodomelatonin binding compared with the control. Injections of ovariectomized (OVX) rats with E2 or P4 alone or in combination for 11 days induced a partial restoration of 2-[125I]iodomelatonin binding in the OVX rats. The results show that mt1 melatonin receptors in the rat antimesometrial stroma are regulated by ovarian hormones.