Pathogenic bacteria of the genus Yersinia harbor a 70-kb plasmid required for virulence. The plasmid-encoded virulence proteins of yersiniae are positively regulated at the transcriptional level by the product of the virF gene, the key activator of the system. virF encodes a DNA-binding protein related to the AraC family of transcriptional activators. The VirF protein from Yersinia enterocolitica is a 30-kDa protein that forms dimers in vitro and that specifically binds to the promoter region of VirF-regulated genes. In this work, we determined the sequences of eight VirF-binding sites from four different genes, by DNase I or hydroxyl radical footprinting. The protected regions, about 40 bases long, were aligned, and a number of conserved residues were identified. A 13-bp sequence resembling TTTTaGYcTtTat (in which nucleotides conserved in > or = 60% of the sequences are in uppercase letters and y indicates C or T) appeared, either isolated or as an inverted repeat in each of the eight sites.