The growth hormone-releasing factor (GRF)-containing neuronal system was immunohistochemically studied in the rat hypothalamus. The immunolabeled cell bodies were determined by intraventricular administration of colchicine 24 h before killing. In intact animals, the neurons appeared in the ventral portion of the arcuate nucleus (group 1) and in the area surrounding the ventromedial nucleus (group 2). Most of the cell bodies also indicated immunoreactivity for tyrosine hydroxylase (TH). The immunoreactive fibers accumulated showing a palisade arrangement in the external layer of the median eminence. The rats treated neonatally with monosodium glutamate revealed group 2 neurons and a few immunoreactive fibers in the median eminence. Half-anterolateral deafferentation of the medial basal hypothalamus, which was performed to isolate group 1 neurons or both group 1 and 2 neurons from the other brain parts, did not remarkably affect the appearance of the fibers in the median eminence. However, the perikarya were hypertrophic and strongly immunolabeled for GRF and TH. It is concluded that the fibers containing GRF in the median eminence derive mostly from group 1 neurons, and that the neurons may be regulated by an inhibitory mechanism by other neurons on the outside of the deafferented hypothalamic islands. GRF synthesized in group 2 neurons may act on other neurons as a neurotransmitter-like substance.