Bone marrow cells taken from mice treated eight days previously with 5-fluorouracil, formed colonies consisting of 10-100 cells after four days of incubation in methylcellulose cultures containing only 500 cells/dish, in the presence of partially purified synergistic factor from human placental-conditioned medium (SFHPlac) and macrophage colony-stimulating factor (CSF-1). Replating of these colonies revealed a high incidence (27%) of another class of high-proliferative-potential colony-forming cells (HPP-CFC) responsive only to the synergistic factor in WEHI-3B-conditioned medium (SFW, which appears to be identical to interleukin 3) plus CSF-1. These colonies contained no HPP-CFC responsive to SFHPlac plus CSF-1, although primary cultures incubated for 14 days in the presence of SFHPlac plus CSF-1 formed large colonies (diameter greater than 0.5 mm), indicating the presence of HPP-CFC responsive to SFHPlac plus CSF-1 in the starting marrow. Primary cultures containing SFW alone, or purified interleukin 3 alone, also gave rise to colonies consisting of 10-100 cells after four days of incubation in methylcellulose cultures; however, the cells from these colonies were unable to form large colonies on replating in the presence of either CSF-1 plus SFHPlac or CSF-1 plus SFW. These results suggest that two distinct populations of HPP-CFC exist and that the population of HPP-CFC stimulated by CSF-1 plus SFHPlac differentiates to form HPP-CFC that respond to CSF-1 plus SFW.