The effect of thyroglobulin (TG) on binding of TSH to thyroid plasma membranes was studied in vitro. Human and bovine thyroid plasma membranes have specific binding sites for bovine [125I]TSH. The binding of [125I]TSH is inhibited by the addition of purified TG (100 ng/microgram/ml). The inhibitory mechanism appears to be noncompetitive when subjected to Lineweaver-Burk analysis. Incubation of TG with TSH did not show an interaction, as assessed by sucrose gradient centrifugation. Plasma membranes prepared from human thyroid tissue have specific binding sites for human TG, as shown by [125I]TG binding assay. The TG binding was not affected by adding low concentrations of unlabeled bovine TSH. In the presence of very high concentrations of TSH, TG binding was increased. Hemoglobin, beta-lactoglobin, and ovalbumin did not have an inhibitory effect on [125I]TSH and [125I] TG binding to membrane preparations. Both [125I]TSH and [125I]TG binding were inhibited by 10 mM neuraminic acid. These results suggested that 1) TG released from thyroid gland may have a regulatory effect on TSH binding to its specific receptor, and 2) there are specific binding sites for TG on the thyroid plasma membrane.