Although there is abundant evidence from animal studies that 1,25-dihydroxychole-calciferol [1,25-(OH)2D3] is synthesized in the kidney, it is still unknown whether human bone might also be involved in the formation of this metabolite. To investigate whether 1,25-(OH)2D3 can be synthesized by human bone, 25-hydroxycholecalciferol (25-OH D3) metabolism was assessed in vitro by incubation with human trabecular bone. The bone tissue, obtained from the femoral heads of 34 patients undergoing hip replacement surgery, was ground and thoroughly cleaned of blood and bone marrow. Five hundred mg of spongiosa were incubated with (3H)-25-OH D3 at a concentration of 1.67 nM in 2 ml Eagle’s minimum essential medium. The metabolites formed were separated either by LH 20 column chromatography followed by high performance liquid chromatography (HPLC) or by HPLC alone and the elution profiles compared to either commercially available or biologically synthesized tritiated 1,25-(OH)2D3 and 24,25-(OH)2D3. The formation of a metabolite which was chromatographically similar to 1,25-(OH)2D3 was observed in 15, and a metabolite with the chromatographic features of 24,25-(OH)2D3 in 13 of 21 bone samples. In 4 samples an additional metabolite more polar than 1,25-(OH)2D3 was observed. I n 6 bone samples no chromatographic evidence for the production of any metabolite of 25-OH D3 could be found. Ten per cent isopropanol, 1 ml of uremic serum or replacement of NADPH by NADH in the incubation medium completely suppressed the production of all derivatives. The demonstration that human bone is able to metabolize 25-OH D3 may be important for the understanding of bone physiology.