The ribA gene, encoding GTP cyclohydrolase II in Escherichia coli, is a member of the soxRS regulon, which is induced by superoxide-generating agents. By evaluating lacZ expression driven by the ribA promoter carrying different lengths of upstream region in a monolysogen, we found that the superoxide-responsive element resides between 56 and 94 nucleotides upstream of the transcriptional start site. Purified SoxS protein bound to this region and protected nucleotides between positions -80 and -58 from degradation by DNase I. This region contains a putative SoxS-binding sequence (soxbox) in reverse orientation. The SoxS protein interacted specifically with four guanine residues within the soxbox sequence, as demonstrated by methylation interference analysis. These results clearly indicate that SoxS binds to the reversed soxbox sequence in the ribA gene, while in other known genes of the soxRS regulon it binds to the normally oriented soxbox. Possible modes of interaction between SoxS and RNA polymerase are discussed.