This study shows for the first time that 1,25-dihyroxyvitamin D₃[1,25-(OH)₂D₃] and two fluorinated analogues of 1,25-(OH)₂D₃ [24,24-F₂-1,25-(OH)₂D₃ and 26, 26, 26, 27, 27, 27-F₆-1,25-(OH)₂D₃] induce macrophage differentiation of human normal and leukemic myeloid stem cells. The addition of either 1,25-(OH)₂D₃ or one of the two fluorinated analogues of 1,25-(OH)₂D₃ at concentrations as low as 10^-9 m to culture plates containing normal human marrow cells stimulated myeloid stem cells to preferentially differentiate to colonies of monocytes and macrophages. Over 80% of the normal human myeloid colonies were composed of only monocytes and macrophages in culture plates containing 10^-7 m 1,25-(OH)₂D₃ or one of the fluorinated analogues. In contrast, control plates not containing 1,25-(OH)₂D₃ and the two vitamin D analogues induced macrophage differentiation of leukemic colony-forming cells taken from patients. In plates containing 10^-7 m 1,25-(OH)₂D₃ or one of the analogues at 10^-6 m, 80% of chronic myelogenous leukemia and approximately 50% of acute nonlymphocytic leukemia colony-forming cells differentiated to macrophage-like cells. In contrast, control plates had about 30 and 20% macrophage colonies in cultures from chronic myelogenous leukemia and acute nonlymphocytic leukemia patients, respectively. Our findings suggest that 1,25-(OH)₂D₃ may play a role in hematopoiesis and that the compound or a related analogue may possibly have a therapeutic role in some leukemias.