There is increasing evidence that an important mechanism by which platelet production is regulated depends upon a humoral substance (thrombopoietin) that affects the production of platelets by megakaryocytes. Plasma from thrombocytopenic donors increases the rate of appearance or concentrations of subsequently administered Na235SO4 or selenomethionine-75Se in platelets. Both isotopes are initially incorporated into the cytoplasm of megakaryocytes, and labeled platelets appear in the circulation after their production and release from megakaryocytes. Thrombopoiesis-stimulating activity also can be detected in the plasma of normal donors when endogenous thrombopoiesis has been suppressed in recipient assay animals by the hypertransfusion of platelets. Recent studies have indicated that certain fractions of plasma from throbocytopenic donors are also capable of stimulating thrombopoiesis in recipient animals. The nature of thrombopoietin(s) and its mechanism of action remain unknown. However, currently available data indicate that thrombopoiesis-stimulating factors may act both on diploid precursors and immature megakaryocytes and upon maturing megakaryocytes. The site of production of thrombopoietin also is unknown. Although the sensor that regulates thrombopoietin or other humoral mediators of thrombopoiesis has not been identified, it appears that platelet numbers, per se, are not the sole variable to which megakaryocytopoiesis eventually responds.