Studies of 3H—TRH (40 Ci/mM) binding to adenohypophyses have been performed in vitro. After a 90 min incubation period, tissues were homogenized in an 0.3M sucrose solution and subjected to differential centrifugation. 3H—TRH binding was more than four—fold greater in the anterior pituitary 10,800 X g pellet than in the corresponding fractions of liver, renal cortex, and myocardium. Binding was enhanced in pituitaries derived from 19 day propylthiouracil treated rats and reduced in rats pre—treated with parenteral L—thyroxin. 3H—TRH binding paralleled the specific activity of the plasma membrane enzyme 5’ nucleotidase. TRH binding could be competitively inhibited by native TRH. Two optical analogues of TRH, with reduced and absent biologic activity respectively, exhibited parallel reductions in their ability to inhibit binding. The time course of TRH binding at 37 C and TRH mediated TSH secretion were similar. It is concluded that the first event in TRH action, analogous to that for larger protein hormones, is selective binding to receptor sites on the plasma membrane of TSH secreting cells. (Endocrinology92: 888, 1973)