Lanosterol was converted to a 14-demethylated metabolite, 4,4-dimethylzymosterol by Saccharomyces cerevisiae microsomes. This metabolism was mediated by a cytochrome P-450 (P-450/14DM). However, a reconstituted system consisting of P-450/14DM and its reductase converted lanosterol to the 14-desaturated derivative of 4,4-dimethylzymosterol, 4,4-dimethyl-5 alpha-cholesta-8, 14,24-trien-3 beta-ol (trienol). When AY-9944 was added to the reaction system with the microsomes, the trienol was formed with corresponding decrease in 4,4-dimethylzymosterol. These observations indicate that the 14 alpha-demethylation of lanosterol by yeast microsomes occurs sequentially via the trienol. Reduction of the trienol to 4,4-dimethylzymosterol is mediated by an AY-9944-sensitive reductase.