The effects of hypophysectomy on thyrotropin releasing hormone (TRH), TRH-glycine (TRH-Gly) and pre-pro-TRH (178-199) concentrations in the rat hypothalamus, cerebrum, cerebellum, brain stem, stomach and retina were studied 7 days after the operation. The hypophysectomized rats were administered a single i.p. injection of T4 (500 μg/kg), T3 (100 μg/kg) or bovine TSH (1.25 IU/kg), and 5 rats of each subgroup were decapitated at 4 hours later. After hypophysectomy, TRH-Gly and pre-pro-TRH (178-199) concentrations in the hypothalamus increased significantly and TRH concentrations decreased after hypophysectomy. After the injection of T4, T3, TRH or TSH TRH-Gly and pre-pro-TRH (178-199) concentration in the hypothalamus of hypophysectomized rats decreased significantly, while that of TRH significantly increased. No changes in TRH, TRH-Gly and prepro-TRH (178-199) concentration in other tissues were observed after hypophysectomy or hormone treatment. The findings suggest that hypophysectomy stimulated TRH synthesis and release in the hypothalamus, that TRH, TSH, T3 and T4 regulate hypothalamic TRH levels, and that proTRH synthesis in the tissues except the hypothalamus may not be regulated by thyroid hormone.