The metabolism of 25-hydroxyvitamin D 3 was studied with a crude mitochondrial cytochrome P450 extract from pig kidney and with recombinant human CYP27A1 (mitochondrial vitamin D 3 25-hydroxylase) and porcine CYP2D25 (microsomal vitamin D 3 25-hydroxylase). The kidney mitochondrial cytochrome P450 catalyzed the formation of 1α,25-dihydroxyvitamin D 3, 24,25-dihydroxyvitamin D 3 and 25,27-dihydroxyvitamin D 3. An additional metabolite that was separated from the other hydroxylated products on HPLC was also formed. The formation of this 25-hydroxyvitamin D 3 metabolite was dependent on NADPH and the mitochondrial electron transferring protein components. A monoclonal antibody directed against purified pig liver CYP27A1 immunoprecipitated the 1α- and 27-hydroxylase activities towards 25-hydroxyvitamin D 3 as well as the formation of the unknown metabolite. These results together with substrate inhibition experiments indicate that CYP27A1 is responsible for the formation of the unknown 25-hydroxyvitamin D 3 metabolite in kidney. Recombinant human CYP27A1 was found to convert 25-hydroxyvitamin D 3 into 1α,25-dihydroxyvitamin D 3, 25,27-dihydroxyvitamin D 3 and a major metabolite with the same retention time on HPLC as that formed by kidney mitochondrial cytochrome P450. Gas chromatography-mass spectrometry (GC-MS) analysis of the unknown enzymatic product revealed it to be a triol different from other known hydroxylated 25-hydroxyvitamin D 3 metabolites such as 1α,25-, 23,25-, 24,25-, 25,26- or 25,27-dihydroxyvitamin D 3. The product had the mass spectrometic properties expected for 4β,25-dihydroxyvitamin D 3. Recombinant porcine CYP2D25 converted 25-hydroxyvitamin D 3 into 1α,25-dihydroxyvitamin D 3 and 25,26-dihydroxyvitamin D 3. It can be concluded that both CYP27A1 and CYP2D25 are able to carry out multiple hydroxylations of 25-hydroxyvitamin D 3.