Understanding the regulation of adhesins defines a pathogenic bacterium's interaction with the local environment within the host. In certain strains of Streptococcus pyogenes, transcription of prtF, the gene which encodes the fibronectin-binding adhesin protein F, is activated by RofA under anaerobic conditions. RofA binds specifically to DNA in its target promoters and autoregulates its own expression. In this study, we have used DNase I protection assays to further investigate the interaction of RofA with its target promoters. In the region between rofA and the gene which encodes protein F (prtF), RofA binds to two distinct sites: a smaller site (17 bp) adjacent to the rofA promoter, and a larger site (40 bp) adjacent to the prtF promoter. Analysis of fusions to a novel reporter gene whose product consists of the fusion of the N-terminal secretion domain of protein F with the C- terminal enzymatic domain of the enterococcal alkaline phosphatase (PhoZ) revealed that the small RofA binding site had no direct role in control of prtF transcription but contributed to regulation of rofA. Comparison in several strains representing different patterns of prtF expression indicated that the larger site was required for activation of rofA and of prtF in all strains by both RofA-dependent and -independent pathways. Thus, it would appear that a common recognition sequence provides separate entries to a final common pathway in S. pyogenes virulence gene expression. The identification of multiple RofA-like proteins and promoters with RofA binding sites implies the existence of a widespread interacting regulatory network.