An isozyme of cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation was purified from human liver using column chromatography, including immunoaffinity chromatography. The purified protein exhibited a single protein band (53 kDa) on sodium dodecylsulfate-polyacrylamide gel electrophoresis. When reconstituted with NADPH-cytochrome P-450 reductase, the purified protein showed an activity of 14 alpha-demethylation of 24,25-dihydrolanosterol (3.20 nmol/min per mg protein). The apparent Km value for 24,25-dihydrolanosterol was found to be 27 microM. This enzyme converted in the reconstituted system, the oxygenated intermediates of 24,25-dihydrolanosterol 14 alpha-demethylation, 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol, to the 32-nor compound, 4,4-dimethylcholesta-8,14-dien-3 beta-ol.