Aerobic growth of Streptococcus pneumoniae results in production of amounts of hydrogen peroxide (H₂O₂) that may exceed 1 mM in the surrounding media. H₂O₂ production by S. pneumoniae has been shown to kill or inhibit the growth of other respiratory tract flora, as well as to have cytotoxic effects on host cells and tissue. The mechanisms allowing S. pneumoniae, a catalase-deficient species, to survive endogenously generated concentrations of H₂O₂ that are sufficient to kill other bacterial species is unknown. In the present study, pyruvate oxidase (SpxB), the enzyme responsible for endogenous H₂O₂ production, was required for survival during exposure to high levels (20 mM) of exogenously added H₂O₂. Pretreatment with H₂O₂ did not increase H₂O₂ resistance in the mutant, suggesting that SpxB activity itself is required, rather than an H₂O₂-inducible pathway. SpxB mutants synthesized 85% less acetyl-phosphate, a potential source of ATP. During H₂O₂ exposure, ATP levels decreased more rapidly in spxB mutants than in wild-type cells, suggesting that the increased killing of spxB mutants was due to more rapid ATP depletion. Together, these data support the hypothesis that S. pneumoniae SpxB contributes to an H₂O₂-resistant energy source that maintains viability during oxidative stress. Thus, SpxB is required for resistance to the toxic by-product of its own activity. Although H₂O₂-dependent hydroxyl radical production and the intracellular concentration of free iron were similar to that of Escherichia coli, killing by H₂O₂ was unaffected by iron chelators, suggesting that S. pneumoniae has a novel mechanism to avoid the toxic effects of the Fenton reaction.