Depriving Saccharomyces cerevisiae strain GL7 of exogenous unsaturated fatty acid supplements causes this sterol biosynthetic mutant to accumulate squalene at the expense of squalene epoxide and squalene diepoxide. To further characterize the apparent relationship between squalene epoxidase activity and membrane fatty acid composition, a variety of unsaturated fatty acids differing in their chain lengths and in the positions and orientation (cis or trans) of their double bonds were tested for their ability to promote turnover of endogenous squalene in cells previously deprived of olefinic supplements. All of the unsaturated fatty acids tested were found to restore squalene epoxidase activity but there were marked differences in their efficacies that best were correlated with the extent to which they reduced the medium chain (C-10 + C-12) saturated fatty acid content of cellular phospholipids. Additional studies demonstrated that de novo protein synthesis was required for the restoration of squalene epoxidase activity in unsaturated fatty acid-deprived cells.