Thrombopoietin (Tpo) is a major regulator of megakaryopoiesis both in vivo and in vitro. Tpo initiates its biologic effects by binding to the Mpl receptor, which is a member of the hematopoietin receptor family. To define the Tpo binding characteristics of the Mpl receptor, we iodinated purified 70-kD recombinant human Tpo using the Bolton-Hunter reagent. Autoradiographic analysis of ¹²⁵I-Tpo binding to normal human marrow mononuclear cells showed many grains specifically associated with megakaryocytes; there were no grains specifically associated with myeloblasts or erythroblasts. Equilibrium binding experiments with ¹²⁵I-Tpo and normal human platelets showed a single class of high-affinity receptors (kd, 190 pmol/L) with approximately 30 Mpl receptors per platelet. Affinity cross-linking with ¹²⁵I-Tpo showed that the Mpl receptor on platelets is of molecular weight ∼98 kD. Despite their sequence similarity, erythropoietin and Tpo did not cross-compete for binding to BaF3 cells engineered to coexpress Mpl receptor and erythropoietin receptor. Progeny of normal human burst-forming units-erythroid (BFU-E) contained Mpl receptor mRNA, and flow cytometric analysis showed the presence of Mpl receptor protein on the surface of these cells. These data indicate that display of the Mpl receptor is not limited to the megakaryocytic lineage, but also includes progeny of BFU-E. Like receptors for other hematopoietic cytokines, the binding affinity of the Mpl receptor for Tpo is high, with relatively few receptors displayed per cell. These results suggest that the effects of Tpo to speed red blood cell recovery after myelosuppressive therapy in vivo and to enhance colony-forming unit-erythroid generation in vitro may be mediated by direct interaction of Tpo and erythroid progenitor cells.