The cell bodies of origin of axons terminating in the median eminence have been identified by retrograde axonal transport of horseradish peroxidase (HRP) or 125I-wheat germ agglutinin (WGA). The tracers were injected into the median eminence by pressure and under direct visual control, using a ventral surgical approach. The retrogradely labeled cells are exclusively located within the hypothalamus. The most heavily labeled cells are parvocellular neurons in the arcuate nucleus, the periventricular area, the medial part of the paraventricular nucleus, and the rostral paraventricular nucleus; a few cells are also located in the rostral part of the preoptic area immediately lateral and dorsal to the organum vasculosum of the lamina terminalis (OVLT). Less heavily located cells are found in the magnocellular neurosecretory nuclei, including the lateral parts of the paraventricular and rostral paraventricular nuclei, the supraoptic nucleus, and the accessory magnocellular nuclei. Retrogradely labeled cells are not found in the ventromedial hypothalamic nucleus, except for a few lightly labeled cells in the posterior division of the nucleus. However, if the injected tracer spreads into the arcuate nucleus, labeled cells are present throughout the ventromedial nucleus. Labeled cells are not found in other parts of the hypothalamus, including lateral and posterior portions of the preoptic area, the anterior hypothalamic area, and the suprachiasmatic nucleus, or in catecholaminergic cell groups of the midbrain, pons, and medulla. Control injections of HRP into the posterior pituitary and the ventromedial nucleus produce patterns of cell labeling which are very distinct from that seen with injections into the median eminence. Following injections into the posterior pituitary, the cells of the magnocellular neurosecretory nuclei are all heavily labeled, but small cells in the parvocellular neuronal groups are not labeled. Direct injections into the ventromedial nucleus resulted in labeled cells in widespread parts of the hypothalamus, as well as in the bed nucleus of the stria terminalis and the lateral septum, in parts of the amygdaloid complex and the subiculum, and in several cell groups in the midbrain, pons, and medulla.