ABSTRACT Saccharomyces cerevisiae , along with other eukaryotes, is resistant to tetracyclines. We found that deletion of SOD1 (encoding Cu/Zn superoxide dismutase) rendered S. cerevisiae hypersensitive to oxytetracycline (OTC): a sod1Δ mutant exhibited a >95% reduction in colony-forming ability at an OTC concentration of 20 μg ml −1 , whereas concentrations of up to 1,000 μg ml −1 had no effect on the growth of the wild type. OTC resistance was restored in the sod1 Δ mutant by complementation with wild-type SOD1 . The effect of OTC appeared to be cytotoxic and was not evident in a ctt1 Δ (cytosolic catalase) mutant or in the presence of tetracycline. SOD1 transcription was not induced by OTC, suggesting that constitutive SOD1 expression is sufficient for wild-type OTC resistance. OTC uptake levels in wild-type and sod1 Δ strains were similar. However, lipid peroxidation and protein oxidation were both enhanced during exposure of the sod1 Δ mutant, but not the wild type, to OTC. We propose that Sod1p protects S. cerevisiae against a mode of OTC action that is dependent on oxidative damage.