The ability of Ralstonia solanacearum to cause disease on plants depends on its type III secretion system (TTSS) encoded by hrp genes. The expression of hrp genes and known TTSS substrates is coordinately regulated by HrpB, a member of the AraC family of transcriptional regulators. Two HrpB-regulated promoters (hrpY and popABC) were characterized by deletion analysis, and the HrpB-dependent activation of these promoters was found to be conferred by a 25-nucleotide DNA element, the hrp_II box (TTCGn16TTCG), which is present in other hrp promoters. The hrp_II box element is an imperfect plant inducible promoter box, an element which was originally found in hrp promoters of Xanthomonas campestris (S. Fenselau and U. Bonas, Mol. Plant-Microbe Interact. 8:845-854, 1995) but which was not characterized at the molecular level. Site-directed mutagenesis showed that the hrp_II box is essential for hrpY promoter activation in vivo. Functional analysis of the hrp_II box element identified critical parameters that are required for HrpB-dependent activity. Further mapping analyses of several other hrpB-dependent promoters also indicated that the position of the hrp_II box is conserved, at −70 to −47 bp from the transcriptional start. As a first step toward identifying novel TTSS effectors, we used the hrp_II box consensus sequence to search for potential HrpB-regulated promoters in the complete genome sequence of R. solanacearum strain GMI1000. Among the 114 genes identified, a subset of promoters was found to have a structural relationship with hrp promoters, thus providing a pool of candidate genes encoding TTSS effectors.