The effect of vitamin B2 on the cellular reduction and cytotoxicity of chromate(VI) was studied using Chinese hamster V-79 cells. Electron spin resonance studies showed that incubation of cells with Na2CrO4 resulted in the formation of both chromium(V) and chromium(III) complex and that cellular pretreatment with riboflavin (Vitamin B2) for 24 h prior to exposure increased the level of chromium(V) complex, but the level of chromium(III) remained unchanged. Analysis of flavin derivatives revealed that pretreatment with vitamin B2 increased free riboflavin without altering flavin adenine dinucleotide and flavin mononucleotide. In addition, the level of the flavoenzyme glutathione reductase, which is capable of reducing chromate, was unaffected by pretreatment with vitamin B2. However, treatment of cells with vitamin B2 and Na2CrO4 augmented the inhibition of glutathione reductase attributable to Na2CrO4 alone. Using a colony-forming assay, pretreatment with vitamin B2 resulted in a decrease of cytotoxicity after exposure to the lethal concentration of chromate (15 microM) but did not affect the cytotoxicity at sublethal concentration of this metal (5-7.5 microM). Alkaline elution studies demonstrated that Na2CrO4 induced alkali-labile sites in the DNA of cells in a concentration-dependent manner (5-15 microM) and pretreatment with vitamin B2 resulted in an increase of these DNA lesions at all concentrations of Na2CrO4. The results, showing that vitamin B2 enhances chromate-induced alkali-labile lesions and chromium inhibition of glutathione reductase, might be due to an increase of chromium(V) species, possibly through its ability to directly reduce chromium(VI). The results also suggest that the extent of DNA lesions induced by chromate may not correlate directly with the cytotoxic effects of this metal.