The biologically active vitamin D3 metabolite 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) has been demonstrated to have differentiative and antiproliferative effects on myeloid tumors of human or murine origin. Its effects on normal murine hemopoiesis were tested by addition of the seco-steroid to long-term bone marrow cultures optimized for either myelopoiesis or B-lymphopoiesis. The addition of 10(-8) M 1,25(OH)2D3, but not 10(-8) M 25(OH)D3, to myeloid bone marrow cultures (MBMC) resulted in a complete cessation of hemopoiesis by 4 weeks, because no hemopoietic cells or colony-forming units were detected. This result was observed whether or not the cultures were initiated and maintained in hydrocortisone. A potential effect of 1,25(OH)2D3 on the production of myeloid growth factors by adherent layer cells in the cultures was examined, but this function was not affected by 1,25(OH)2D3 treatment. Further, adherent layers that had been treated with 1,25(OH)2D3 for 3 weeks were capable of supporting myelopoiesis upon seeding with a stromal cell-depleted population of bone marrow cells. Transfer of MBMC to lymphoid bone marrow culture (LBMC) conditions results in the cessation of myelopoiesis and the initiation of B-cell production. Lymphopoiesis did not initiate in 1,25(OH)2D3-pretreated MBMC that were transferred to LBMC conditions, indicating that the pool of B-cell precursors present in MBMC had been depleted by exposure of MBMC to 1,25(OH)2D3. When 1,25(OH)2D3 (10(-8) M) was added to MBMC at the time of transfer to LBMC conditions, the seco-steroid did not affect induction of B-lymphopoiesis, although the overall cellularity was less in 1,25(OH)2D3-treated cultures than in control cultures.