The morphological substrate for the central mechanisms that control growth hormone (GH) release in the rat hypothalamus was investigated immunohistochemically by light and electron microscopy. In electron-microscopic studies, a dual immunolabeling technique was employed to demonstrate pairs of peptides, i.e. rat hypothalamic growth hormone-releasing factor (rhGRF) and somatostatin (SRIH), rhGRF and substance P (SP), and rhGRF and methionine-enkephalin-Arg6-Gly7-Leu8 (Enk-8), in different neuronal structures. Immunoreactivity of rhGRF was detected as silver-gold particles and those of the other substances as diaminobenzidine products by preembedding immunostaining procedures. In the external layer of the median eminence, axonal terminals immunolabeled for rhGRF and for SRIH showed the same pattern of distribution and close proximity. The neuronal inputs to GRF cell bodies in the arcuate nucleus were examined, and SRIH, SP and Enk-8 fibers with varicosities were found to form dense networks around the perikarya of GRF neurons, suggesting the presence of synaptic associations. Axonal terminals immunolabeled for SRIH, SP or Enk-8, and unlabeled terminals appeared to form coincidental synaptic junctions on GRF perikarya. These findings suggest that the central regulation of GH release occurs at the levels of the median eminence and the cell bodies.