1,25-Dihydroxyvitamin D-3 (1,25(OH)2D3) suppresses colony formation of normal human granulocyte macrophage progenitors (CFU-GM) and induces differentiation of colonies into monocyte macrophages in vitro. We examined whether or not the target cell of 1,25(OH)2D3 is only CFU-GM in the suppression and differentiation of colonies by the agent. Reduction of colony counts was observed only when 1,25(OH)2D3 was added to CFU-GM cultures at days 0 or 3, and after day 5 the agent did not affect the colony count. However, the delayed addition of 1,25(OH)2D3 elevated the proportion of granulocyte macrophage (GM) or macrophage (M) colonies even after 5 days of culture. We confirmed transformation of day 11 colony cells into macrophages by 1,25(OH)2D3 during the following 3 days' culture period based on serial observations of single colonies. Day 11 colonies contained very few CFU-GM and their most immature cells were promyelocytes. Short-term exposure to 1,25(OH)2D3 (4 h) of bone marrow cells, which had been precultured for 24 to 72 h with a purified granulocyte-colony stimulating factor caused a reduction of colony counts but did not elevate the proportions of GM or M colonies. These results indicate that 1,25(OH)2D3 inhibits the growth of CFU-GM itself and induces differentiation into macrophages at the progeny level (probably promyelocytes) and not at the level of CFU-GM.