The effects of recombinant cytokines on the ploidy of human megakaryocytes derived from megakaryocyte progenitors were studied using serum-free agar cultures. Nonadherent and T cell-depleted marrow cells were cultured for 14 days. Megakaryocyte colonies were identified in situ by the alkaline phosphatase anti-alkaline phosphatase technique, using monoclonal antibody against platelet IIb/IIIa. The ploidy of individual megakaryocytes in colonies was determined by microfluorometry with DAPI (4',6-diamidino-2-phenylindole) staining. Recombinant human interleukin 3 (rhIL-3) and recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) supported megakaryocyte colony formation in a dose-dependent manner. However, both rhIL-3 and rhGM-CSF had no definite ability to increase the ploidy values. Recombinant human erythropoietin (rhEpo) or recombinant human macrophage colony-stimulating factor (rhM-CSF) by itself did not stimulate the growth of megakaryocyte progenitors. rhEpo or rhM-CSF, however, stimulated increases in the number, size and ploidy values of megakaryocyte colonies in the presence of rhIL-3 or rhGM-CSF. Recombinant human interleukin 6 (rhIL-6) showed no capacity to generate or enhance megakaryocyte colony formation when added to the culture alone or in combination with rhIL-3. rhIL-6, however, increased the ploidy values in colonies when added with rhIL-3. These results show that rhEpo, rhM-CSF and rhIL-6 affect endomitosis and that two factors are required for megakaryocyte development.