BACKGROUND: Data from several studies support the hypothesis that thrombopoietin (TPO) plasma levels are regulated via circulating platelet (PLT) numbers by binding to PLT TPO receptors (TPO‐Rs). In this study, PLT numbers and TPO plasma levels were measured following the transfusion of unmanipulated, sham‐saturated, and TPO‐R‐saturated PLT preparations to provide additional in vivo evidence for this regulatory mechanism. STUDY DESIGN AND METHODS: Following in vitro experiments to characterize pegylated recombinant human megakaryocyte growth and development factor (PEG‐rHuMGDF) binding characteristics, PLT numbers and TPO plasma levels were measured following the transfusion of unmanipulated, sham‐saturated, and TPO‐R‐saturated PLT preparations in thrombocytopenic patients. Sham‐saturated and TPO‐R‐saturated PLTs were prepared by a 1‐hour incubation without and with 40 ng per mL of PEG‐rHuMGDF, respectively, and subsequent washing and resuspension. RESULTS: In vitro, 2.72 ± 0.8 ng of PEG‐rHuMGDF per 1 × 108 PLTs was bound within 1 hour of incubation. No additional PEG‐rHuMGDF was bound following a second incubation with PEG‐rHuMGDF, and bound PEG‐rHuMGDF was not released over time. In vivo, TPO plasma levels decreased significantly (p < 0.001), by 30.7 ± 5.8 and 20.9 ± 2.1 percent after transfusion of unmanipulated and sham‐saturated PLT preparations, respectively. However, TPO plasma levels were unaffected after the transfusion of TPO‐R‐saturated PLTs despite comparable transfusion‐induced PLT count increases. CONCLUSION: These data strongly support the concept that binding to PLT TPO‐R is directly involved in human TPO plasma level regulation in vivo.