Ubiquinone is an essential redox component of the aerobic respiratory chains of bacteria and mitochondria. It is well established that mammalian ubiquinone can function in its reduced form (ubiquinol) as a lipid-soluble antioxidant preventing lipid peroxidation. The objective of this study was to test the hypothesis that prokaryotic ubiquinone is involved in the defence against oxidative stress in the cytoplasmic membrane. The rate of superoxide production by rapidly respiring wild-type Escherichia coli membranes was twofold higher than in the slowly respiring membranes from a ubiCA knockout mutant. However, large amounts of superoxide accumulated in the Ubi- membranes compared to wild-type membranes, which possess superoxide-scavenging ubiquinol. Likewise, the rate of H2O2 production was twofold higher in the wild-type, but the overall production of H2O2 was again significantly higher in the Ubi- membranes. Inclusion of a water-soluble ubiquinone homologue (UQ-1) effectively decreased the amount of H2O2 produced in the Ubi- membranes in a concentration-dependent manner. Addition of UQ-2 to the membranes was even more effective in limiting accumulation of H2O2 than was UQ-1, suggesting a role for the side-chain in conferring liposolubility in the antioxidative defence mechanism. Intracellular H2O2 concentration was increased 1.8-fold in the ubiCA mutant, and expression of the katG gene, encoding the catalase hydroperoxidase I, as well as catalase enzyme activity, were increased twofold in this mutant. The ubiCA mutant was hypersensitive to oxidative stress mediated by CuSO4 or H2O2; sensitivity to the latter could be abolished by addition of cysteine. This phenotype was also exhibited by a ubiG mutant, defective in the last step of UQ biosynthesis and therefore expected to accumulate several UQ biosynthetic intermediates. These observations support the participation of reduced ubiquinone as an antioxidant in E. coli. The ubiCA mutant exhibited a pleiotropic phenotype, being resistant to heat, linolenic acid and phleomycin. Resistance to the two latter compounds is probably due to reduced uptake. Like mutants unable to synthesize the quinol oxidase, cytochrome bd, the ubiCA mutant was also sensitive to dithiothreitol, an effect that is attributed to inability of the respiratory chain to maintain an appropriate redox balance in the periplasm.