GH is an anabolic hormone that is essential for normal linear growth and has important metabolic effects throughout life. The ultradian rhythm of GH secretion is generated by the intricate patterned release of two hypothalamic hormones, somatostatin (SRIF) and GHRH, acting both at the level of the pituitary gland and within the central nervous system. The recent discovery of ghrelin, a novel GH-releasing peptide identified as the endogenous ligand for the GH secretagogue receptor and shown to induce a positive energy balance, suggests the existence of an additional neuroendocrine pathway for GH control. To further understand how ghrelin interacts with the classical GHRH/SRIF neuronal system in GH regulation, we used a combined physiological and histochemical approach. Our physiological studies of the effects of ghrelin on spontaneous pulsatile GH secretion in conscious, free-moving male rats demonstrate that 1) ghrelin, administered either systemically or centrally, exerts potent, time-dependent GH-releasing activity under physiological conditions; 2) ghrelin is a functional antagonist of SRIF, but its GH-releasing activity at the pituitary level is not dependent on inhibiting endogenous SRIF release; 3) SRIF antagonizes the action of ghrelin at the level of the pituitary gland; and 4) the GH response to ghrelin in vivo requires an intact endogenous GHRH system. Our dual chromogenic and autoradiographic in situ hybridization experiments provide anatomical evidence that ghrelin may directly modulate GHRH mRNA- and neuropeptide Y mRNA-containing neurons in the hypothalamic arcuate nucleus, but that SRIF mRNA-expressing cells are not major direct targets for ghrelin. Together, these findings support the idea that ghrelin may be a critical hormonal signal of nutritional status to the GH neuroendocrine axis serving to integrate energy balance and the growth process.