Estrogen exerts important feedback effects upon the biosynthetic and secretory behavior of gonadotropin-releasing hormone (GnRH) neurons to control reproductive functioning. The mechanism of estrogen action upon these neurons is unclear and seems likely to involve the transsynaptic regulation of GnRH neurons. The objective of the present study was to identify the estrogen-receptive neural populations which project to the general vicinity of the GnRH perikarya in the rostral preoptic area and diagonal band of Broca (rPOA/DBB) of the ewe. Intact breeding-season ewes received an injection of the retrograde tracer fluorogold (FG) into the rPOA/DBB, and their hypothalami and brainstems examined for the presence of FG and estrogen receptor alpha (ERalpha) immunocytochemistry. Retrogradely labeled neurons were identified principally within the lateral septum (LS), lamina terminalis, bed nucleus of the stria terminalis, POA, arcuate nucleus (ARN), ventromedial nucleus (VMN) and median eminence. Smaller numbers of FG-immonoreactive cells were found in the caudal brainstem where they resided mostly in the ventrolateral medulla (VLM). Dual-labeled cells exhibiting both FG and ERalpha staining were prominent in the POA, LS and at all rostrocaudal levels of the VMN and ARN. Small numbers of dual-labeled cells were found in the VLM. These observations indicate that a number of distinct ERalpha-expressing neural populations project to the rPOA/DBB where the majority of the GnRH perikarya are found in the ewe. Although it is not possible to determine the direct connectivity of these projections with GnRH neurons, the findings provide an initial neuroanatomical framework through which the transsynaptic actions of estrogen on ovine GnRH neurons may be tested.