Control of 125-dihydroxyvitamin D (1,25-(OH)2D) synthesis is believed to be primarily at the level of expression of the vitamin D-1-hydroxylase (CYP1alpha; CYP1alpha) gene. Once transcribed, generation of product, as catalyzed by 1-hydroxylase, depends upon the availability of various co-factors, molecular oxygen, electrons as well as substrate to the enzyme. Here we provide evidence that the quantity of product 1,25-(OH)2D generated also relies on the presence and level of expression of the intracellular vitamin D binding protein-1 (IDBP-1) and its capacity to promote 24-hydroxylase (CYP24) gene expression. Stable transfection of the IDBP-1 cDNA increased 1,25-(OH)2D synthesis up to 700% (p < 0.001) in cells devoid of 24-hydroxylating potential but only 70% (p = 0.018) in cells in which the CYP24 gene is expressed. IDBP-1-mediated increase in 1,25-(OH)2D production was independent of any change in CYP1alpha expression but highly dependent on the ability of exogenously-added or endogenously-synthesized 1,25-(OH)2D to stimulate CYP24 gene expression. These data suggest that IDBP-1 is capable of controlling 1,25-(OH)2D production by modulating the delivery of 1) substrate 25-OHD to in the mitochondrial CYP1alpha gene product and 2) CYP1alpha product 1,25-(OH)2D to the vitamin D receptor for upregulation of expression of the catabolic CYP24 gene.