Summary Incubation of osteoblast-like cells with [3H]25-(OH)D3 and varying bovine serum albumin (BSA) concentrations resulted in a dramatic change in the accumulation of 1,25-(OH)2D3 and 24,25-(OH)2D3 in the medium. At 0.1% BSA 1,25-(OH)2D3 formation was transient and 24,25-(OH)2D3 was the main product after 3 h. At 2% BSA accumulation of 1,25-(OH)2D3 was sustained whereas 24,25-(OH)2D3 formation was suppressed. At low BSA levels added [3H]1,25-(OH)2D3 was rapidly metabolized to 1,24,25-(OH)3D3 and more polar metabolites. The effect of increasing BSA concentrations on 25-(OH)D3 metabolism was mimicked by addition of cycloheximide. This indicates that high BSA levels prevent the induction of 24-hydroxylase activity in this system, probably by lowering of the free 25-(OH)D3 concentration. The accumulation of 1,25-(OH)2D3 from 25-(OH)D3 not only depends on the 1α-hydroxylase activity, but also on the further metabolism of 1,25-(OH)2D3 by 24-hydroxylation.