Peptide-like materials which release thyrotropin (TSH) from the pituitary in vivo and in vitro were obtained from the hypothalamus of the ox, sheep and pig and from human neurohypophysial tissue. Thyrotropic hormone releasing factor (TRF) activity was measured in vitro by the isolated rat pituitary incubation method of Saffran and Schally. TSH secreted into the medium was assayed by the release of 13II from the thyroid gland of the mouse as described by McKenzie and by the uptake of 131I by the tadpole's thyroid as described by Bowers. Addition of TRF preparations to the incubation media significantly increased the release of TSH from the pituitaries. TSH release in vitro was related to the dose of TRF preparations from all 4 species. One TRF preparation was active in vitro at a dose of 10 μg. TRF activity was also measured in vivo in the rat. TRF preparations from each species increased plasma TSH levels in thyroidectomized, triiodothyronine-treated rats. These studies support the concept that the hypothalamus takes part in the regulation of TSH release from the anterior pituitary gland. (Endocrinology77: 609,1965)