Pituitary gland growth hormone (GH) secretion is influenced by two hypothalamic neuropeptides: growth hormone-releasing hormone (GHRH) and somatostatin. Recent data also suggest that estrogen modulates GH release, particularly at the time of the preovulatory luteinizing hormone surge, when a coincident surge of GH is observed in sheep. The GHRH neurons do not possess estrogen receptor alpha (ERalpha), suggesting that estrogen does not act directly on GHRH neurons. Similarly, few somatotropes express ERalpha, suggesting a weak pituitary effect of estradiol on GH. It was hypothesized, therefore, that estradiol may affect somatostatin neurons to modulate GH release from the pituitary. Using immunocytochemical approaches, the present study revealed that although somatostatin neurons were located in several hypothalamic sites, only those in the arcuate nucleus (13% +/- 2%) and ventromedial nucleus (VMN; 29% +/- 1%) expressed ERalpha. In addition, we found that all neurons immunoreactive for somatostatin-14 were also immunoreactive for somatostatin-28(1-12). To determine whether increased GH secretion in response to estradiol is through modulation of GHRH and/or somatostatin neuronal activity, a final study investigated whether c-fos expression increased in somatostatin- and GHRH-immunoreactive cells at the time of the estradiol-induced LH surge in intact anestrous ewes. Estradiol significantly (P < 0.05) increased the percentage of GHRH (estradiol, 75% +/- 3%; no estradiol, 19% +/- 2%) neurons expressing c-fos in the hypothalamus. The percentage of somatostatin-immunoreactive neurons coexpressing c-fos in the estradiol-treated animals was significantly (P < 0.05) higher (periventricular, 44% +/- 3%; arcuate, 72% +/- 5%; VMN, 81% +/- 5%) than in the control animals (periventricular, 22% +/- 1%; arcuate, 29% +/- 3%; VMN, 31% +/- 3%). The present study suggests that estradiol modulates the activity of GHRH and somatostatin neurons but that this effect is most likely mediated through an indirect interneuronal pathway.