Separate regions of the anterior periventricular area (PVA), median eminence (ME) and frontal cortex from brains of hypophysectomized female and intact male rats were extracted and homogenized. The LH-RH content of these homogenates was determined by a specific double antibody radioimmunoassay. LH-RH was found in the PVA as well as in the ME of both hypophysectomized female and intact male rats; it was not detected in the frontal cortex. Extracellular recordings were made from neurons in the PVA and 55 neurons were activated antidromically by stimulating the ME. This suggests that PVA neurons exist with axons terminating in the ME. It is assumed that these axons are unmyelinated since their conduction velocity was approximately 0.3-0.8 m/sec. In addition to the antidromically-activated neurons, 26 neurons with characteristics of transsynaptic activation were recorded in the PVA following stimulation of the ME. Some of the units recorded from the ME-arcuate (ARC) region were also activated either antidromically or transsynaptically by stimulating the PVA. The effect of PVA stimulation on LH release was tested in ovariectomized, estrogen-treated rats with or without anterior deafferentation of the hypothalamus. Increased LH concentration in serum was elicited by PVA stimulation of rats in which deafferentation was either not performed or not completed; no increase in serum LH level was observed following PVA stimulation in rats with complete deafferentation. These data suggest that PVA neurons synthesize LH-RH, which is released at the ME.