Saccharomyces cerevisiae GL7 cells require exogenous sterol and unsaturated fatty acid for growth. When grown in the presence of cholesterol or 7-dehydrocholesterol, the cells incorporated less saturated fatty acid into phospholipids than cells grown with ergosterol, stigmasterol, or beta-sitosterol as the sterol source. This lower saturated fatty acid content was most pronounced in phosphatidylethanolamine, slightly less so in phosphatidylcholine, and least evident in phosphatidylserine and phosphatidylinositol. Growing the cells with the various sterols did not affect the ratios of individual phospholipids. The ability of strain GL7 to use 7-dehydrocholesterol as the only sterol supplement for growth was dependent upon the nature of the unsaturated fatty acids added to the growth medium. In the presence of linoleic, linolenic, or a mixture of palmitoleic and oleic acids, excellent growth was observed with either ergosterol, cholesterol, or 7-dehydrocholesterol. However, when the medium was supplemented with either oleic or petroselenic acid, the cells grew more slowly (oleic) or much more poorly (petroselenic) with 7-dehydrocholesterol than with ergosterol. A specific relationship between sterol structure and membrane fatty acid composition in yeast cells is implied.