The effect of hydrogen peroxide onTreponema pallidumwas investigated. Thein vitroloss of virulence (as measured by rabbit inoculation) ofT.pallidumwas accelerated by as low as 100 μMhydrogen peroxide in the complex maintenance medium used. Higher doses led to rapidly accelerated death with 500 μMhydrogen peroxide causing sterilization of the medium within 3 to 4 h. Since hydrogen peroxide is known to cause single-stranded breaks in DNA, the effect of hydrogen peroxide on the treponemal genome was examined. Extensive breakage was caused by 100 μMhydrogen peroxide as determined on alkaline sucrose gradients. A limit was reached at 250 μMand above. Single-stranded breaks could be demonstrated as early as 5 – 10 min after exposure to hydrogen peroxide when the treponemes were exposed to 250 μMhydrogen peroxide; accelerated death was evident by 2 h past exposure demonstrating that DNA breakage was preceding death. Treponemal death caused by penicillin did not result in DNA breakage. The repair-proficient bacteriumEscherichia coliK-12 was compared withT.pallidum. It required 10 – 100 times more hydrogen peroxide to cause various levels of breakage.Escherichia coliK-12 rapidly repaired DNA breakage once hydrogen peroxide was removed by addition of catalase.Treponema pallidum, in comparison, showed little or no repairin vitro. Addition of catalase or dithiothreitol to the medium protected against all but a low level of breakage; this may reflect on the ability of catalase and reducing agents to protectT.pallidumagainst oxygen toxicityin vitro.