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Human marrow cells were processed sequentially by density centrifugation and by velocity sedimentation in serum-free Percoll gradients in order to purify megakaryocytes and to determine if these cells are the source of the growth factor derived from platelets. Cell homogenates were made from the resulting fractions and tested for growth-promoting activity(ies) in 3T3 cells and in well characterized human marrow fibroblasts. Growth was evaluated by 3H-TdR incorporation and changes in DNA cell content, as measured by flow microfluorometry. The highest mitogenic activity was derived from homogenates of low density (less than 1.050 g/cu cm), rapidly sedimenting cells. This fraction contained the highest percentage of megakaryocytes. The assessment of growth-promoting activity(ies) derived from various megakaryocyte-enriched marrow cell homogenates containing different proportions of megakaryocytes demonstrated a positive correlation between the number of megakaryocytes and their stimulatory capacity as determined by 3H-TdR uptake. The growth-promoting activities elicited from homogenates of platelets and marrow fractions enriched for megakaryocytes were similar. The dose--response curves for both were parallel, and they were both temperature resistant and trypsin sensitive. These findings implicate megakaryocytes as a source of the growth factor derived from platelets and suggest that megakaryocytes may play a role in the pathogenesis of the marrow fibrosis observed in myeloproliferative disorders by stimulating fibroblast proliferation and collagen secretion.