The effect of partially purified thrombopoietic stimulatory factor (TSF) on megakaryocytopoiesis was studied using the soft-gel colony-forming assay and a short-term marrow liquid culture system (STLC) and compared to the effects of megakaryocyte colony-stimulating activity present in pokeweed mitogen-stimulated spleen cell-conditioned medium (PWCM). Nonadherent cells from STLC were sampled daily for acetylcholinesterase-positive cells and megakaryocyte progenitor cells (CFU-M). CFU-M were assayed in the soft-gel colony-forming system using PWCM as a source of colony-stimulating activity. Proliferative capacity of CFU-M obtained from liquid culture was determined from megakaryocyte colony size (number of megakaryocytes per colony) following plating of cells in a secondary colony-forming assay. Megakaryocytes were grouped into four maturation classes and megakaryocyte diameter was determined on acetylcholinesterase-stained cytocentrifuged cells using an eye-piece micrometer. TSF produced no CFU-M-derived colonies in the soft-gel colony-forming assay. Addition of TSF to STLC had no effect on the total number of CFU-M, megakaryocyte colony size, or total number of megakaryocytes compared to unstimulated STLC. However, on days 4-9 there was a significant increase in megakaryocyte diameter and the proportion of mature (stage III, IV) megakaryocytes obtained from TSF containing STLC compared to unstimulated STLC. In contrast, 5 days after addition of PWCM to STLC a sixfold increase in the total number of CFU-M per flask and a threefold increase in megakaryocytes was observed compared to unstimulated STLC. However, megakaryocyte colony size and megakaryocyte size were significantly reduced and a greater number of immature (stage I, II) megakaryocytes were present in STLC containing PWCM compared to unstimulated STLC. These results indicate that TSF accelerates the maturation of megakaryocytes in vitro and that a factor or factors present in spleen cell-conditioned medium, in addition to influencing megakaryocyte progenitor cell proliferation, also affect (s) megakaryocyte size.