Analysis of the pneumococcal genome sequences from strains R6 and TIGR4 identified a putative alkylhydroperoxidase homologue RT-PCR showed this gene to be expressed in an operon with the downstream open reading frame. No probable function for this second gene is suggested although it appears to be an integral membrane protein. An allelic replacement mutant lacking this two-gene operon in strain D39 was attenuated in competitive infections with the wild type parent. This operon is, therefore, a novel pneumococcal virulence determinant. In line with a role in the response to oxidative stress, this mutant showed enhanced resistance to killing by hydrogen peroxide, a phenotype shared by alkylhydroperoxidase mutants in other bacterial species. The analysis of non-polar single mutants shows that both genes contribute to these phenotypes. Finally, an important role in pneumococcal biology is suggested by the presence of this operon in all 20 clinical isolates examined and the highly conserved sequence of the two genes.