Activation of vitamin D requires hepatic 25-hydroxylation and renal 1alpha-hydroxylation. Defects in renal P450c1alpha are well-described, but few patients with defective vitamin D 25-hydroxylation are reported. The cytochrome P450 enzymes CYP2D6 and CYP27 are potential 25-hydroxylases. We sequenced both genes in two reported families with hepatic 25-hydroxylase deficiency and found no mutations. 25-Hydroxylation occurs in both mitochondria and microsomes. The existence genes encoding distinct enzymes would provide genetic redundancy, explaining the rarity of apparent vitamin D 25-hydroxylase deficiency.