Thrombopoietin (TPO) is a major regulator of platelet production. The concentration of circulating TPO seems to be determined by its binding and internalization by megakaryocytes and platelets. To elucidate the platelet compartments involved in TPO metabolism, we investigated intraplatelet TPO by post-embedding immunoelectron microscopy, incubated platelets with recombinant human (rh)TPO coupled to colloidal gold and visualized the TPO uptake using electron microscopy. TPO concentrations were measured in 12 platelet concentrates (PC) before and after stimulation with thrombin and after disruption of platelets by freezing-thawing. In resting platelets, immunogold labelling revealed a prevailing cytoplasmic localization of TPO antigen and minor labelling within the surface-connected canalicular system (SCCS); storage granules were devoid of labelling. In tracer experiments, TPO-gold was observed on the plasma and SCCS membranes and within the cytoplasm. Upon thrombin stimulation, endogenous TPO was still detected within the cytoplasm by immunolabelling, and tracer experiments revealed TPO-gold within the cytoplasm and on fibrin fibres. After thrombin stimulation of PC, the plasma TPO levels increased to an average of 535%, and after platelet lysis to an average of 1625% compared with plasma values in unstimulated PC. We conclude that platelets contain releasable immunoreactive TPO within the SCCS and within their cytoplasm, but not within granular compartments. Stored immunoreactive TPO is released upon thrombin stimulation, but only to a minor degree.