The effects of prolactin and other hormones applied electrophoretically to 400 neurons in the brain were studied in rats under urethane anesthesia. 51 prolactin-activated neurons were distributed mainly in the nucleus dorsomedialis, the upper part of the nucleus ventromedialis, and the nucleus habenulae. 26 prolactin-inhibited neurons were diffusely distributed from the nucleus arcuatus to the zona incerta. Prolactin failed to affect the neurons of the cerebral cortex. Almost all units recorded in the preoptic and lateral hypothalamic areas were not affected. About half of both prolactin-activated and -inhibited neurons were suppressed by estrogen and betamethasone but were not affected by either oxytocin or thyrotropin releasing hormone. This study affords direct evidence for the presence of prolactin-sensitive neurons in the hypothalamus.