PURPOSE: To prepare an NAD(P)-dependent cholesterol dehydrogenase, by culturing microorganisms belonging to Nocardia genus, etc. and capable of growing under aerobic conditions.

CONSTITUTION: Microorganism belonging to Nocardia genus, Alcaligenes genus, or Proteus genus, and capable of growing under aerobic conditions, e.g. Nocardia sp. No.Ch2-1 (FERM-P No.6217), Alcaligenes sp. No.4 (FERM-P No.6216), Proteus vulgaris IAM 1025, etc. is cultured in a medium containing cholesterol under aerobic conditions, and the objective NAD(P)-dependent cholesterol dehydrogenase is separated from the cultured cells and the culture liquid.