Induction of hematopoietic differentiation was investigated in human promyelocytic leukemia cells [HL-60] using two lipophilic vitamins, retinoic acid and 1 alpha,25-dihydroxyvitamin D3 [1 alpha,25(OH)2D3]. Both vitamins suppressed proliferation and induced differentiation of HL-60 cells, but 1 alpha,25(OH)2D3 was 70- to 100-fold more potent than was retinoic acid on a molar basis. Simultaneous treatment with suboptimal concentrations of 1 alpha,25(OH)2D3 (0.12 to 1.2 nM) and retinoic acid (10 to 100 nM) showed additive effects in reducing nitroblue tetrazolium, a common marker for monocyte-macrophage and granulocyte differentiation. For the study of alternative differentiation of the cells by the two vitamins, we used monoclonal antibodies specific for either human monocyte-macrophages or granulocytes and other markers specific for macrophage differentiation such as alpha-naphthyl acetate esterase activity and adherence to the dish surface. HL-60 cells were induced to differentiate alternatively into macrophages by 1 alpha,25(OH)2D3 or into granulocytes by retinoic acid. When HL-60 cells were treated with various concentrations of 1 alpha,25(OH)2D3 (1.2 to 120 nM) in the presence of 1000 nM retinoic acid which is a concentration sufficient to induce maximal granulocyte differentiation, the appearance of the markers for monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 was not at all affected by the retinoic acid. These results indicate that 1 alpha,25(OH)2D3 and retinoic acid have additive effects in inducing differentiation of HL-60 cells, but monocyte-macrophage differentiation by 1 alpha,25(OH)2D3 occurs much more readily than does granulocyte differentiation by retinoic acid.