Incubation of Chinese hamster V-79 cells with Na2CrO4 plus vitamin B2 resulted in an increase of Na2CrO4-induced DNA single strand breaks. Electron spin resonance (ESR) studies showed that vitamin B2 enhanced the formation of both hydroxyl radical and tetraperoxochromate (V) during the reaction of Na2CrO4 with hydrogen peroxide. Furthermore, ESR studies demonstrated that a chromium (V) species with a g value of 1.977 was formed by the reaction of Na2CrO4 with vitamin B2. These results indicate that chromate reacts with vitamin B2 to form chromium (V) species and also suggest that the enhancement effect of vitamin B2 on chromate-induced DNA single strand breaks may result from an increase of chromium (V)-related hydroxyl radical formation.