Bone cells respond to 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) for mineral mobilization and contain receptors for 1,25(OH)2D3. We report here the expression of 25-hydroxyvitamin D3 (25 (OH)D3) metabolizing enzymes in primary cultures of human bone cells, as well as n a human osteosarcoma cell line. Human bone cells were obtained by enzyme digestion of the extracellular matrix of bone from iliac crest biopsies from 3 male patients without primary bone disease. These cells were plated (5 X 10(4)/min) in medium with 10% fetal calf serum and proliferated to confluence in 10-14 days. At confluence, the medium was replaced with serum-free medium. The cells were preincubated in this serum-free medium for 24 h prior to incubating them 2-4 h with [3H]25(OH)D3 (10-20 nM). The vitamin D metabolites synthesized during this incubation were extracted from the medium and cells with dichloromethane, then separated by chromatography on Sephadex LH-20, followed by high performance liquid chromatography. The cells synthesized 1,25(OH)2D3 and 24,25-dihydroxyvitamin D3 (24,25(OH)2D3) with the specific activities of the 1- and 24-hydroxylases similar in magnitude to those in kidney cells in vitro. The enzymes could be regulated by external perturbations, in that the activity of the 1-hydroxylase was inhibited by preincubation of the cells for 8 h with 1,25(OH)2D3 (10 nM), whereas the 24-hydroxylase was enhanced. Incubation of the cells in a low calcium medium (0.6 mM) depressed the 24-hydroxylase activity. We conclude: 1) normal human bone cells can produce 1,25(OH)2D3 and 24,25(OH)2D3 in vitro in amounts similar to kidney cells, suggesting a physiological significance and 2) this synthesis could account for the increase in osteoclast number in anephric patients with renal osteodystrophy.