OLE1 of Saccharomyces cerevisiae encodes the sole and essential -9 desaturase catalyzing the conversion of saturated to unsaturated fatty acids. Upon ectopic overexpression of OLE1 in S. cerevisiae, significant increases in the membrane oleic acid content were observed. OLE1-overexpressing strains displayed enhanced tolerance to various stresses, better proton efflux, lower membrane permeability, and lessened internal hydrogen peroxide content. The OLE1-mediated enhanced stress tolerance was considerably diminished upon deletion of HOG1, which encodes the mitogen-activated protein kinase (MAPK) Hog1 of the high osmolarity glycerol (HOG) pathway. Furthermore, OLE1 overexpression constitutively activated Hog1, which remained in the cytoplasm. Hog1 activation was accomplished through the MAPK kinase kinase (MAPKKK) Ssk2, but not Ste11 and Ssk22, the other MAPKKKs of the HOG pathway. Despite its cytoplasmic location, activated Hog1 was able to activate the expression of its canonical targets, including CTT1, HSP12, and STL1, and further, the cAMP and stress response elements present in the promoter. OLE1 overexpression neither caused nor relieved endoplasmic reticulum stress. Individually or in combination, the physiological and molecular changes caused by OLE1 overexpression may contribute to enhanced tolerance to various types of stress. Biotechnol. Bioeng. 2017;114: 620-631. (c) 2016 Wiley Periodicals, Inc.