1,25-dihydroxyvitamin D3 [1,25(OH)2D3] has been shown to promote the in vitro differentiation of established human monocytic cell lines and normal human bone marrow progenitor cells toward the macrophage phenotype. The possibility that 1,25(OH)2D3 exerts a similar effect on the differentiation of peripheral monocytes from normal individuals, has been examined in the present study. Monocytes were isolated by density gradient sedimentation on Ficoll-Paque followed by adherence to plastic. Cells were subsequently maintained in culture in 10% autologous serum for 4 weeks either with or without 1,25(OH)2D3 (10(-8) M). In five experiments using monocytes from different donors we found that cells grown in the absence of the hormone underwent morphologic changes toward the macrophage phenotype as well as a gradual increase in the activities of lysosomal enzymes (beta-acetyl-glucosaminidase and beta-galactosidase). In the presence of the hormone the morphologic changes appeared at earlier stages of the culture, and the increase in enzymatic activities occurred earlier and was one- to twofold greater than the activity of the control cells. In addition, 1,25(OH)2D3 enhanced the adherence of the cultured cells.