To investigate the role of hydrogen peroxide in Cr(VI) toxicity in vivo toward bacterial cells, we examined the effect of Cr(VI), hydrogen peroxide, sodium azide, and mannitol on the viability of Escherichia coli. Bacterial cells were incubated for 1 h with shaking in the presence of Cr(VI), hydrogen peroxide, sodium azide as catalase inhibitor, and/or mannitol as radical scavenger. The colony-forming ability and double-strand DNA degradation were examined. The viability assays revealed that Cr(VI) toxicity depended on hydroxyl radicals generated in the reaction involving hydrogen peroxide and chromium. Moreover, incubation of E. coli cells with 10 mM Cr(VI) and 3 mM hydrogen peroxide caused the degradation of double-strand DNA in vivo, which was suppressed by the addition of mannitol. These results indicated that hydroxyl radicals generated in the incubation degraded DNA of E. coli cells, resulting in cell death. In the absence of added hydrogen peroxide, the intracellular concentration of hydrogen peroxide in E. coli was low (below 1 microM). A catalase-defective strain incubated in the absence of added hydrogen peroxide remained fully viable after 1 h but showed decreased viability after prolonged incubation (4-8 h). The addition of mannitol suppressed this decrease, suggesting that hydroxyl radicals may be involved in the expression of Cr(VI) toxicity even without added hydrogen peroxide.