The cDNA for vitamin D 25-hydroxylase in rat liver mitochondria was transfected in COS cells in order to confirm our previous postulation that both 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha-triol 27-hydroxylation and vitamin D 25-hydroxylation are catalyzed by a common enzyme. As a result it was found that both enzyme activities could be reconstituted from the solubilized extract of mitochondria of these cells, NADPH, NADPH-adrenodoxin reductase and adrenodoxin, giving unequivocal evidence that the two enzyme activities are catalyzed by a common enzyme.