Cultures of Saccharomyces cerevisiae strain GL7 auxotrophic for sterol were incubated with a series of sterols and sterol-like molecules (tetracyclic and pentacyclic triterpenoids) in order to determine the structural requirements of sterols for bulk membrane function. For growth support, the 3β-OH group could not be replaced by H, OMe, OBu, NH2, NHOH, OAc, keto, or 3α-OH. A methyl group at C-14 was neither deleterious nor essential for activity. Removal of the C-4 geminal methyl group was obligatory for activity. Thus, no sterol-like triterpenoid supported growth (e.g., tetrahymanol, lanosterol, and cycloartenol). Growth support required a sterol with the longest methylene segment extending from C-20 not to exceed six contiguous C-atoms and the stereochemistry must be C-20 R. No significance could be attributed to branching at C-20 (i.e., to C-21), C-24 (when alkylated), or C-25 (regarding the isopropyl group). Double bonds in the nucleus were not essential for activity since cholestanol supported growth. In several incubations, the addition of trace levels of dietary ergosterol (0.5 μg/ml) to the medium was necessary to promote growth and transformation of the bulk sterol to a membrane competent sterol(s).