We investigated the expression of three platelet alpha-granule proteins, thrombospondin (TSP), von Willebrand factor (vWF), and fibrinogen (Fbg), in a human megakaryocytic cell line (CMK 11-5) by immunocytochemical staining, using the alkaline phosphatase anti-alkaline phosphatase (APAAP) complex method and immunoelectron microscopy of ultrathin-frozen sections. CMK 11-5, established from a Down's syndrome patient with acute megakaryoblastic leukemia, has characteristics closely resembling those of normal megakaryocytes. Under basal conditions, TSP expression was observed in the alpha-granules, whereas the other two proteins were not detected. When the cells were cultured with 12-o-tetradecanoylphorbol-13-acetate (TPA), the expression of TSP was enhanced and vWF was also detected, but not Fbg. Cells cultured in the presence of plasma for 24 h took up Fbg and stored it in their alpha-granules. These findings suggest that TSP and vWF are synthesized by CMK 11-5 cells, with the former being produced more rapidly than the latter, whereas Fbg is endocytosed from the plasma.