A gene having the activity of a hydroxylase that can convert the hydrogen atom at the 25-position of a vitamin D into a hydroxyl group is obtained from the strain of an actinomyces of the genus (Nocardia) or (Amycolata). A 25-hydroxylated vitamin D can be prepared efficiently in a high yield by transforming (Streptomyces lividans) or (Streptomyces griseus) with a plasmid bearing the above gene and treating a vitamin D having a hydrogen atom at the 25-position with the resultant transformant.
