The distribution of 125I-melatonin binding sites in 6 discrete brain regions of female rats at the estrous stage and the effects of ovariectomy and subsequent 17β-estradiol treatment on these binding sites were studied. Specific binding of 125I-melatonin was found in the hypothalamus, medulla-pons, hippocampus, cerebellum, striatum and parietal cortex of the female rats. Ovariectomy produced a large estradiol-reversible decrease in 125I-melatonin binding in the medulla-pons and hypothalamus. In contrast, 125I-melatonin binding sites in the other brain regions were generally unaffected by ovariectomy or estradiol. The estradiol-regulated changes in melatonin binding in the hypothalamus and medulla-pons may reflect the role of a specific estradiol-melatonin interaction in the coordination of the neuroendocrine reproductive axis.