Increased ethanol intake, a major predictor for the development of alcohol use disorders, is facilitated by the development of tolerance to both the aversive and pleasurable effects of the drug. The molecular mechanisms underlying ethanol tolerance development are complex and are not yet well understood.To identify genetic mechanisms that contribute to ethanol tolerance, we examined the time course of gene expression changes elicited by a single sedating dose of ethanol in Drosophila, and completed a behavioral survey of strains harboring mutations in ethanol-regulated genes.Enrichment for genes in metabolism, nucleic acid binding, olfaction, regulation of signal transduction, and stress suggests that these biological processes are coordinately affected by ethanol exposure. We also detected a coordinate up-regulation of genes in the Toll and Imd innate immunity signal transduction pathways. A multi-study comparison revealed a small set of genes showing similar regulation, including increased expression of 3 genes for serine biosynthesis. A survey of Drosophila strains harboring mutations in ethanol-regulated genes for ethanol sensitivity and tolerance phenotypes revealed roles for serine biosynthesis, olfaction, transcriptional regulation, immunity, and metabolism. Flies harboring deletions of the genes encoding the olfactory co-receptor Or83b or the sirtuin Sir2 showed marked changes in the development of ethanol tolerance.Our findings implicate novel roles for these genes in regulating ethanol behavioral responses.