The ontogenesis of growth hormone releasing hormone (GH-RH) containing neurons in the rat hypothalamus has been studied by immunohistochemistry, using a specific anti-rat GH-RH serum. Immunoreactive fibers were first detected in the prospective median eminence on day 18 of gestation. During the subsequent 3 days, they rapidly increased in distribution and intensity of staining within this structure. On day 21, positive fibers were also visible in a plexus within the arcuate nucleus. In 1-day-old rats treated with colchicine, positive perikarya were distributed in several hypothalamic nuclei, including the arcuate nucleus, dorsomedial nucleus, basal lateral hypothalamus, and perifornical region. The distribution was similar to that previously described in adult rats. The intensity of staining in the various hypothalamic regions increased during early postnatal life to levels nearly comparable to those in adult rats by 30 days. These findings showing the early appearance of GH-RH-positive terminals in the median eminence and the wide distribution of the perikarya at an early stage of postnatal life support the view that hypothalamic GH-RH serves an important role in the regulation of growth hormone secretion during late prenatal and early neonatal periods.