An accurate assay method of 25-hydroxyvitamin D3 24-hydroxylase (24-hydroxylase) was established. Kidney mitochondria prepared from vitamin D-replete rats were treated with polyoxyethylenesorbitan monolaurate. The solubilized suspension was ultracentrifuged at 100,000g for 60 minutes and an aliquot of the supernatant was incubated under the saturating concentrations of substrate NADPH and the mitochondrial-type electron transferring proteins, adrenodoxin and NADPH-adrenodoxin reductase. Products were analyzed by high-performance liquid chromatography (HPLC) monitoring effluents at a wavelength of 265 nm. The maximal velocity of the enzyme in vitamin D-replete rats was 400 pmol/minute per mg of protein, which was considerably higher than those reported by previous authors who used intact kidney mitochondria as the enzyme source. In applying the new assay method, an interesting property was found; Michaelis constant of 24-hydroxylase for 25-hydroxyvitamin D3 [25(OH)D3] was 0.6 μM, which was 35-fold lower than that for 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] which was 20.9 μM. This fact indicates that affinity of the enzyme to 25(OH)D3 is 35-fold higher than that to 1α,25(OH)2D3. These data suggest that 25(OH)D3 is the preferred substrate to 1α,25(OH)2D3.