We investigated possible cross talk between endogenous antioxidants glutathione, spermidine, and glutathionylspermidine and drug efflux in Escherichia coli. We found that cells lacking either spermidine or glutathione are less susceptible than the wild type to novobiocin and certain aminoglycosides. In contrast, exogenous glutathione protects against both bactericidal and bacteriostatic antibiotics. The glutathione protection does not require the AcrAB efflux pump but fails in cells lacking TolC because exogenous glutathione is toxic to these cells.