Corticotropin‐releasing hormone (CRH) is the major regulator of the pituitary‐adrenal axis. CRH‐immunoreactive perikarya are widely distributed in the central nervous system; however, only those which participate directly in the regulation of adrenocorticotropin are connected to the portal circulation in the external zone of the median eminence. The present study describes the identification of these hypophysiotropic neurons using retrograde labeling and CRH immunocytochemistry. Fuoro‐Gold was injected peripherally then, 5 days later, the animals were treated with colchicine. Twenty‐four hours later the animals were sacrificed, and their brains were immunostained for CRH with the indirect immunofluorescence technique. The results indicate that the vast majority of the Fluoro‐Gold‐accumulating and CRH‐immunopositive perikarya (hypophysiotropic neurons) are located in the medial parvicellular subdivision of the paraventricular nucleus (PVN). However, not each CRH‐immunoreactive neuron contains Fluoro‐Gold, i.e. a small portion of these neurons project to areas of the brain other than the median eminence. The anterior, lateral and periventricular subdivisions of the PVN also contain hypophysiotropic CRH‐immunoreactive perikarya, however, their number is much less than in the medial parvicellular subdivision. Scattered double‐labeled cells are also present in the medial preoptic area and the dorsal hypothalamus, just behind the PVN. These results support previous observations that the PVN, particularly the medial parvicellular subdivision, is the predominant source of the hypophysiotropic CRH neurons.