We examined colony formation by mouse spleen cells in a serum-free culture system containing purified erythropoietin (Ep), interleukin-3 (IL-3) or a combination of the two factors. Purified Ep supported erythroid colony and burst formation but did not support formation of other types of colonies. IL-3 alone did not support erythroid burst formation in culture. However, IL-3 significantly increased the number and size of erythroid bursts. IL-3 but not Ep supported megakaryocyte colony formation. Ep did not enhance megakaryocyte colony formation or megakaryocyte populations in multilineage colonies supported by IL-3.