Interleukin 3 (IL3) promotes the proliferation of murine hematopoietic progenitor cells, including the megakaryocytic colony-forming cell (CFU- MK). To determine whether IL3 influences more differentiated cells in the murine megakaryocytic lineage, IL3 was added to microcultures of single megakaryocytes isolated from CFU-MK-derived colonies. After two days of culture, cell diameter and DNA content were analyzed. Eighty- five percent of cells initially between 12 and 20 micron in diameter increased in size in response to IL3, while only 12% of these cells increased in size in its absence. The percentage of cells responding to IL3 varied inversely with the size of the initial cells. The culture procedure did not alter the relatively linear relationship between size and ploidy, suggesting that increments in cell size are accompanied by increased ploidy. After three days of culture, acetylcholinesterase (AchE) activity was assayed. A small but significant increment in AchE activity was observed in the presence of IL3. The data show that, at least in vitro, IL3 is a differentiation-promoting factor of the murine megakaryocytic lineage.