Reactive oxygen species (ROS) are potent oxidants that attack chromosomal DNA and free nucleotides, leading to oxidative DNA damage that causes genetic alterations. To avoid the ROS-mediated mutagenesis, cells have elaborate mechanisms including powerful antioxidant components and repair pathways that eliminate oxidative DNA damage. Because of the effective anti-mutagenic functions, it has been unclear to what extent the ROS contribute to spontaneous mutagenesis. Here we show that a significant portion of spontaneous mutations is actually caused by the ROS in aerobically growing Escherichia coli cells. Using the rpsL gene as a mutational target sequence, we established an experimental procedure to analyze spontaneous mutations occurring under a strictly anaerobic condition. Strong mutator phenotypes of cells defective in both mutM and mutY genes or ones lacking mutT gene were completely suppressed under the anaerobic condition, indicative of an absence of hydroxyl radicals in the cells. From a series of analyses with wild-type E. coli cells grown under different redox conditions, it appeared that 89% of base substitutions were caused by the ROS, especially hydroxyl radicals, in cells growing in the atmosphere. The ROS-mediated spontaneous mutations included highly site-specific base substitutions, two types of randomly occurring transversions, G:C[RIGHTWARDS ARROW]C:G and A:T[RIGHTWARDS ARROW]T:A, and –1 frameshifts at non-iterated base sequences.