In the rat median eminence immunoreactive galanin nerve fibers and terminals are present in high numbers in the external layer, and fibers in moderate numbers are seen in the internal layer. The possible sources of these galanin-containing fibers were studied by means of radioimmunoassay and immunohistochemistry in rats with different types of hypothalamic lesions. Galanin-like neurons were found both (1) in the magnocellular hypothalamo-neurohypophyseal system and (2) in the parvocellular hypothalamo-median eminence-anterior pituitary system. Cell bodies containing galanin-like immunoreactivity were localized in the supraoptic, magnocellular paraventricular and accessory magnocellular neurons with axons traversing the internal layer and terminating in the posterior pituitary. Surgical isolation of these neurons from the median eminence resulted in a marked depletion of immunoreactive galanin from the internal layer of the median eminence and the posterior pituitary. Due to the retrograde accumulation of axonally transported substances in cells proximal to the lesions, immunoreactive galanin-like cells became visible in the supraoptic and paraventricular nuclei ipsilateral to the knife cuts, and levels of galanin-like immunoreactivity increased in these nuclei 7 days after bilateral transections of the hypothalamo-hypophyseal tract. Immunoreactive galanin fibers in the external layer of the median eminence around the portal capillaries were found to be of paraventricular and arcuate nucleus origin. Bilateral paraventricular lesions caused marked (70%) reduction in levels of galanin-like immunoreactivity in the median eminence. The remaining 30% of the galanin immunoreactivity in the external layer may arise from the arcuate nucleus, which contains a great number of galanin-containing cell bodies. Thus, galanin-like immunoreactivity accumulated retrogradely in parvocellular paraventricular and arcuate nucleus neurons after transections of their fibers in the lateral retrochiasmatic area or in the median eminence, respectively.