Cells whose nuclei accumulated 3H-estradiol were identified autoradiographically in fixed, frozen sections of colchicine-treated rat hypothalamus (n = 3 animals). After autoradiogramm development, these sections were subjected to immunocytochemistry using rabbit antirat prolactin antiserum and the avidin-biotinylated horseradish peroxidase method. In the hypothalamus, a substantial subset of the neurons containing immunoreactive prolactin accumulated 3H-estradiol in their nuclei: of 3,642 immunoreactive cells examined, 1,216 had autoradiographically labeled nuclei, or about 33%. The immunoreactive prolactin neurons with autoradiographically labeled nuclei were located in the medial basal hypothalamus intermingled with immunoreactive prolactin neurons whose nuclei were not labeled autoradiographically. Since hypothalamic immunoreactive prolactin neurons have a rich and widely distributed fiber system, the present results suggest that estrogen, acting through a subset of these neurons, can modify directly the neuronal activity of several brain regions which regulate diverse aspects of the reproductive effort. Also, since immonoreactive prolactin and immunoreactive β-endorphin exist in the same hypothalamic cell population, opioid peptides derived from pro-opiomelanocortin may mediate some effects of estrogen on the neural circuitry regulating reproduction