Sensitive in vitro lanosterol 14 alpha- and 4 alpha-methylsterol oxidase assays, particularly suitable for cell extracts of tissue culture cells, were developed and validated. Using these assays, we showed that the biochemical lesion of mutant 215, a cholesterol-requiring Chinese hamster ovary cell auxotroph isolated and partially characterized previously [Chang, T. Y., Telakowski, C., Vanden Heuvel, W., Alberts, A. W., & Vagelos, P. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 832-836], was localized at the 4 alpha-methylsterol oxidase enzyme system. The defect in 4 alpha-methylsterol oxidase activity in mutant 215 cells could be demonstrated by using either 4,4-dimethylcholestanol or 4 alpha-methylcholestanol as the substrate, suggesting that the enzyme systems responsible for 4 alpha-methyl- and 4,4-dimethylsterols may share a common component. However, demethylation of the C-14 alpha methyl group was found to occur at identical rates in wild-type and mutant 215, suggesting that C-14 alpha demethylation and C-4 alpha demethylation may occur by separate enzyme systems. A [3H]dihydrolanosterol incorporation experiment in intact cells of wild-type and mutant 215 supported these conclusions. Despite these results, a [14C]acetate pulse experiment indicated that [14C]lanosterol, instead of its 14C-labeled 14-demethylated sterol derivative(s), accumulated in intact cells of mutant 215. Possible implications of these findings for the mechanisms of lanosterol demethylation reactions are discussed.