Expression of the Pseudomonas aeruginosa type III secretion system (T3SS) is activated by ExsA, a member of the AraC/XylS family of transcriptional regulators. In the present study we examine the DNA-binding properties of ExsA. ExsA was purified as a histidine-tagged fusion protein (ExsA(His)) and found to be monomeric in solution. ExsA(His) specifically bound T3SS promoters with high affinity as determined by electrophoretic mobility shift assays (EMSA). For each promoter tested two distinct ExsA-DNA complexes were detected. Biochemical analyses indicate that the higher-mobility complex consists of a single ExsA(His) molecule bound to DNA while the lower-mobility complex results from the binding of two ExsA(His) molecules. DNase I protection assays demonstrate that the ExsA(His) binding site overlaps the -35 RNA polymerase binding site and extends upstream an additional approximately 34 bp. An alignment of all 10 ExsA-dependent promoters revealed a number of highly conserved nucleotides within the footprinted region. We find that most of the highly conserved nucleotides are required for transcription in vivo; EMSA-binding assays confirm that several of these nucleotides are essential determinants of ExsA(His) binding. The combined data support a model in which two ExsA(His) molecules bind adjacent sites on the promoter to activate T3SS gene transcription.