Cytochrome P-450(14DM) catalyzing lanosterol 14 alpha-demethylation was purified from rat liver and was characterized by its catalytic properties and mode of expression. The purified protein, with an apparent molecular mass of 51 kDa, showed 24,25-dihydrolanosterol 14 alpha-demethylation activity (3.05 nmol/min/mg protein) in a reconstituted system with an apparent Km value of 40 microM. The reconstituted enzyme converted 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol (the oxygenated intermediates of 24,25-dihydrolanosterol) to 4,4-dimethylcholesta-8,14-dien-3 beta-ol (the 32-nor compound). Immunoblot analysis of liver microsomes from male and female rats showed that the enzyme protein was expressed at an early stage of development and attained its maximum at 4 weeks after birth, retaining the same level thereafter. No difference was evident between males and females in the level of cytochrome P-450(14DM) in the liver up to 7 weeks after birth.