To investigate the function of the EvgA response regulator, we compared the genome-wide transcription profile of EvgA-overexpressing and EvgA-lacking Escherichia coli strains by oligonucleotide microarrays. The microarray measurements allowed the identification of at least 37 EvgA-activated genes, including acid resistance-related genes gadABC and hdeAB, efflux pump genes yhiUV and emrK, and 21 genes with unknown function. EvgA overexpression conferred acid resistance to exponentially growing cells. This acid resistance was abolished by deletion of ydeP, ydeO, or yhiE, which was induced by EvgA overexpression. These results suggest that ydeP, ydeO, and yhiE are novel genes related to acid resistance and that EvgA regulates several acid resistance genes. Furthermore, the deletion of yhiE completely abolished acid resistance in stationary-phase cells, suggesting that YhiE plays a critical role in stationary-phase acid resistance. The multidrug resistance in an acrB deletion mutant caused by EvgA overexpression was completely abolished by deletion of yhiUV, while the emrKY deletion had no effect on the increase in resistance by EvgA overexpression. In addition, EvgA overexpression did not confer resistance in a tolC-deficient strain. These results suggest that YhiUV induced by EvgA overexpression is functionally associated with TolC and contributes to multidrug resistance.