Megakaryocytes develop from diploid precursor cells that, after variable numbers of mitoses, cease cell division and then undergo synchronous nuclear endoreduplication (endomitosis). Megakaryocyte colony formation represents an in-vitro model of these processes in which the number and ploidy of colony cells reflect the activity of the mitotic and endomitotic pathways, respectively. We have analyzed the size and ploidization of murine megakaryocyte colonies grown in agar and examined the influence of interleukin-3 (IL-3) on these parameters. Colonies were identified in situ by staining for acetylcholinesterase and the ploidy of colony cells was determined by fluorescence cytophotometry. More than 98% of the megakaryocytes that developed in culture could be analyzed. In cultures of unfractionated marrow cells stimulated by either pokeweed mitogen-stimulated spleen cell-conditioned medium (PWM-SCM, a crude source of megakaryocyte colony-stimulating activity) or IL-3, the modal ploidy of day-5 colony megakaryocytes was 16N (range 2N-128N). The distribution of colony size was described by an inverse exponential relationship. Colony size and geometric mean ploidy were inversely correlated under conditions of maximal stimulation with PWM-SCM and at all concentrations of IL-3 tested. Increasing concentrations of IL-3 in cultures of either unfractionated marrow cells or nonadherent T-lymphocyte-depleted (NATD) marrow cells stimulated similar dose-dependent increases in the mean size and numbers of megakaryocyte colonies but did not significantly alter their ploidy distribution. However, the mean ploidy of colony megakaryocytes in IL-3-stimulated cultures of NATD marrow cells was significantly less (P less than 0.001) than the mean ploidy of megakaryocytes in IL-3-stimulated cultures of unfractionated marrow cells. The mean ploidy of megakaryocytes, which developed in PWM-SCM-stimulated cultures, was not affected by initial accessory cell depletion. We conclude that the size and ploidy characteristics of day-5 murine megakaryocyte colonies are structured as continua and that IL-3 stimulates an increase in mean colony size and numbers without affecting ploidization. T-lymphocytes and adherent cells elaborate an activity which promotes endomitosis in vitro; factors in PWM-SCM can substitute for this activity.