ABSTRACT Several regulators within the AraC family control the expression of genes known or thought to be required for virulence of bacterial pathogens. One of these, Rns, activates transcription from an unprecedented variety of binding-site locations. Although nearly all prokaryotic activators bind within a small region upstream and adjacent to the promoter that they regulate, Rns does not bind within this region to activate its own promoter, P rns . Instead, to activate P rns , Rns requires one binding site 224.5 bp upstream and one downstream of the transcription start site. We show in this study that several other AraC family activators recognize the same binding sites as Rns and share with it the ability to utilize a downstream binding site. Like Rns, other members of this group of activators positively regulate the expression of virulence factors in pathogenic bacteria. These regulators are also able to activate transcription from promoter-proximal upstream binding sites since they are able to substitute for Rns at P coo , a promoter with only upstream binding sites. Thus, Rns is the prototype for a group of regulators, which include CfaR, VirF, AggR, and CsvR and which activate transcription from locations that are more diverse than those of any other known activator.