Oxidoreductases such as glutaredoxin are a major class of enzymes that reversibly catalyze thiol-disulfide exchange reactions. Transfection experiments using geneticin (G418) selection to identify the specific protein S-thiolated substrates of glutaredoxin-1 (Grx-1) noted the curious phenomenon that nontransfected control cells treated with G418 had increased levels of Grx-1 expression. Varied concentrations of gentamicin, kanamycin, and hygromycin increased Grx-1 expression in a time- and dose-dependent fashion in human cultured retinal pigment epithelial cells. Reactive oxygen species formation after aminoglycoside exposure correlated directly to aminoglycoside treatment. Further indication that oxidation regulates Grx-1 expression was noted by the positive effect of phorbol 12-myristate 13-acetate, a known inducer of redox-sensitive AP-1 transcription factor. In agreement with this hypothesis was the finding that the physiologic reductant N-acetylcysteine decreased Grx-1 expression whereas tert-butyl hydroperoxide increased Grx-1 expression. Our data suggest that aminoglycosides increased Grx-1 expression in response to oxidative stress.