TRH is present throughout the central nervous system possessing many different functions. Only TRH synthesized in the hypothalamic PVN and transported to the ME regulates anterior pituitary secretion of thyrotropin. To investigate this hypophysiotropic system, we have developed a method using dissected rat PVN and ME, representing mostly cell bodies and nerve terminals respectively, of the same TRH system. Tissues were incubated for four 30 min periods each in Locke's medium with alternatively normal and high KCl concentration. Repeated KCl-induced membrane depolarization resulted in significantly increased TRH release from both tissues (15% of TRH content) which was dependent on Ca2+ influx. Some important differences were found in the regulation of TRH in the PVN and ME: Comparison of TRH content in the tissue before and after incubation with the amount of secreted TRH showed new production of TRH during incubation in the ME, but not in the PVN. Frequent medium replacement during depolarization revealed that TRH secretion at the level of the ME, but not the PVN, is probably inhibited by some substance released during incubation. These data suggest that there is a different regulation of TRH in the PVN and ME and that TRH secreted by the isolated PVN may come mostly from the perikarya and represent paracrine neurohormone secretion.