Intravenous injection of the synthetic tripeptide (PyroGlu-His-Pro-NH2: TRH) effected the prompt release of TSH and prolactin (PRL) from the pituitary of the goitrous rat. Plasma TSH and PRL levels increased 2-3-fold within 1 min after the injection of 0.4 and 2 µg TRH. Intravenous injection of 20 µg of L-thyroxine (T4) induced repletion of TSH to supranormal levels in the adenohypophysis of goitrous rats without a significant change in PRL stores. The acute administration of TRH (2 and 50 µg) to rats after pituitary TSH rebound resulted in a simultaneous increase in circulating levels of the pituitary hormones; this was correlated with the prompt and vigorous extrusion of secretory granules from the pituitary cells. PRL content of the pituitary increased. A relationship was found in rats between the amount of TRH ingested in drinking water and plasma levels of PRL and TSH; hormonal stores in the adenohypophysis usually declined. Ingestion of large amounts of TRH (1,700 µg daily for 8 and 14 days) by the euthyroid rat resulted in a 2–3-fold elevation of the plasma TSH level. In PTU(propylthiouracil)-treated rats ingesting approximately the same amount of TRH, a plasma TSH increase failed to occur. The oral ingestion of TRH for 22–27 days by goitrous, TSH-rebounded rats resulted in a significant diminution in the circulating levels of TSH and PRL, and in ultrastructural manifestations suggestive of impaired release by the adenohypophysis. It is concluded that the acute administration of TRH causes the rapid release of TSH and PRL from the pituitary of the chronically hypothyroid rat. The intensity of the response to TRH is enhanced after pituitary TSH rebound, and synthesis of PRL appears to be augmented. Chronic oral administration of TRH to the goitrous rat results in a diminished release of the pituitary hormones, despite ample stores in th