Streptococcus pneumoniae is an aerotolerant gram-positive bacterium that causes an array of diseases, including pneumonia, otitis media, and meningitis. During aerobic growth, S. pneumoniae produces high levels of H(2)O(2). Since S. pneumoniae lacks catalase, the question of how it controls H(2)O(2) levels is of critical importance. The psa locus encodes an ABC Mn(2+)-permease complex (psaBCA) and a putative thiol peroxidase, tpxD. This study shows that tpxD encodes a functional thiol peroxidase involved in the adjustment of H(2)O(2) homeostasis in the cell. Kinetic experiments showed that recombinant TpxD removed H(2)O(2) efficiently. However, in vivo experiments revealed that TpxD detoxifies only a fraction of the H(2)O(2) generated by the pneumococcus. Mass spectrometry analysis demonstrated that TpxD Cys(58) undergoes selective oxidation in vivo, under conditions where H(2)O(2) is formed, confirming the thiol peroxidase activity. Levels of TpxD expression and synthesis in vitro were significantly increased in cells grown under aerobic versus anaerobic conditions. The challenge with D39 and TIGR4 with H(2)O(2) resulted in tpxD upregulation, while psaBCA expression was oppositely affected. However, the challenge of ΔtpxD mutants with H(2)O(2) did not affect psaBCA, implying that TpxD is involved in the regulation of the psa operon, in addition to its scavenging activity. Virulence studies demonstrated a notable difference in the survival time of mice infected intranasally with D39 compared to that of mice infected intranasally with D39ΔtpxD. However, when bacteria were administered directly into the blood, this difference disappeared. The findings of this study suggest that TpxD constitutes a component of the organism's fundamental strategy to fine-tune cellular processes in response to H(2)O(2).