Sterols modified at various positions of the tetracyclic nucleus were tested as growth supplements for Saccharomyces cerevisiae strain GL7 erg12 heme3. Derivatives of 3 beta-cholestanol or delta 7-3 beta-cholestenol bearing either a single alpha-oriented methyl group of a gem-dimethyl group at C-4 supported the growth of the mutant whereas 4 beta-methyl sterols did not. The nutritionally active alkyl derivatives were metabolized to 4-demethyl sterols while 4 beta-methyl derivatives were incorporated unchanged, indicating that the C-4 demethylase of yeast is specific for alpha-oriented methyl groups. It appears that 4-demethyl sterols are obligatory for growth of this organism. C-4 methyl derivatives of cholesterol did not support growth, suggesting that the delta 5 double bond blocks demethylation at the adjacent C-4. In other experiments, 14 alpha-methyl sterols were effective growth supplements, while 3 alpha-methylcholesterol was totally inactive. Removal of the C-19 methyl group of cholesterol (19-noncholesterol) rendered the sterol somewhat less effective as a sterol source. The sterol specificity for yeast appears to be particularly strict with regard to substituents that add bulk to the A ring of the steroid nucleus.