1,25-Dihydroxyvitamin D3 [1,25-(OH)2D3] has been shown to be a potent agent for monocyte/macrophage differentiation in leukemic cell lines. This study examines the differentiational effects of 1,25-(OH)2D3 in authentic murine bone marrow immature uncommitted precursor cells collected by pretreatment of murine bone marrow with 5-fluorouracil (5-FU). Early precursor cells, collected 1 day after 5-FU treatment, are characterized by low expression of the colony-stimulating factor-1. (CSF-1) receptor and dependence on both CSF-1 and interleukin-1 for growth. Intermediate precursors were collected 5 days after 5-FU treatment and required either CSF-1 or interleukin-3 for growth. Intermediate precursors expressed relatively higher levels of the CSF-1 receptor. Addition of 1,25-(OH)2D3 to these populations inhibited proliferation, as measured by [3H]thymidine incorporation and soft agar colony assay. Furthermore, 1,25-(OH)2D3 caused a more rapid appearance of the CSF-1 receptor in early precursor cells in a dose-dependent metabolite-specific manner. Conversely, CSF-1 receptor expression was decreased in intermediate precursors treated with the steroid. This decrease in receptor expression was also dose dependent and metabolite specific. These findings demonstrate that 1,25-(OH)2D3 1) targets to authentic bone marrow macrophages at various stages of differentiation and 2) modulates expression of the CSF-1 receptor, a protein which, in turn, regulates monocytic maturation.