The arcuate nucleus/ventromedial hypothalamic nucleus (ARC/VMH) region is thought to relay estrogen feedback signals to gonadotropin-releasing hormone (GnRH) cells in the sheep brain. This region sends major projections to the lateral preoptic area (lPOA), ventral bed nucleus of the stria terminals (vBnST) and the ventro-caudal division of the median preoptic nucleus (vcMePON) with little direct input to GnRH cell bodies, suggesting interneuronal relay to GnRH neurons. The brain stem also provides input to the POA. The present study aimed to identify possible relay circuits in the POA and BnST to GnRH neurons. Biotinylated dextran amine (BDA) was injected into lPOA (n=6), vBnST (n=2), vcMePON (n=3) and periventricular nucleus (PeriV; n=1) of ewes for anterograde tracing. GnRH immunoreactive (IR) perikarya appearing to receive input from BDA-containing varicosities were identified by fluorescence microscopy, with further analysis by confocal microscopy. When BDA was injected into rostral and caudal regions of lPOA (n=3), no tracer-filled varicose fibers were found in contact with GnRH-IR perikarya. Injections into the center of the lPOA (n=3) indicated direct projections to GnRH-IR cells. Injections into the vBnST, vcMePON and PeriV indicated that cells of these regions also provide input to GnRH cells. BDA-containing varicosities found in the MPOA were immunoreactive for NPY or were GABAergic or glutamatergic when the tracer was injected into vBnST and lPOA, but not when injections were placed in the vcMePON. With injection into the PeriV, tracer-filled varicosities in the MPOA were not immunoreactive for somatostatin or enkephalin. Injection of FluoroGold into ventral POA retrogradely labeled cells in the above mentioned areas, but few were also immunoreactive for estrogen receptor-α. Thus, cells of the vBnST, lPOA, vcMePON and PeriV project to GnRH neurons. These cells may provide an interneuronal route to GnRH neurons from the ARC/VMH, the brain stem and other regions of the brain.