Abstract: The understanding of the molecular basis of yeast resistance to ethanol may guide the design of rational strategies to increase process performance in industrial alcoholic fermentations. In this study, the yeast disruptome was screened for mutants with differential susceptibility towards stress induced by high ethanol concentrations in minimal growth medium. Over 250 determinants of resistance to ethanol were identified. The most significant GO terms enriched in this dataset are those associated to intracellular organization, biogenesis and transport, in particular regarding the vacuole, the peroxisome, the endosome and the cytoskeleton, and those associated to the transcriptional machinery. Clustering the proteins encoded by the identified determinants of ethanol resistance by their known physical and genetic interactions highlighted the importance of the vacuolar protein sorting machinery, the V-ATPase complex and the peroxisome protein import machinery. Evidence was obtained showing that vacuolar acidification and increased resistance to the cell wall lytic enzyme beta-glucanase occur in response to ethanol induced stress. Based on the genome-wide results, the particular role of the FPS1 gene, encoding a plasma membrane aquaglyceroporin which mediates controlled glycerol efflux, in ethanol stress resistance was further investigated. FPS1 expression contributes to decreased (3)H-ethanol accumulation in yeast cells, suggesting that Fps1p may also play a role in maintaining ethanol intracellular level during active fermentation. The increased expression of FPS1 confirmed the important role of this gene in alcoholic fermentation leading to an increased final ethanol concentration under conditions that lead to high ethanol production.