Exotoxin A production in Pseudomonas aeruginosa is regulated positively or negatively by several genes. Two such regulatory genes, ptxR and ptxS, which are divergently transcribed from each other, have been described previously. While computer analysis suggested that the ptxR-ptxS intergenic region contains potential binding sites for several regulatory proteins, the mechanism that regulates the expression of either ptxR or ptxS in P. aeruginosa is not known. The presence of a P. aeruginosa protein complex that specifically binds to a segment within this region was determined. In this study the binding region was localized to a 150 bp fragment of the intergenic region and the proteins that constitute the binding complex were characterized as P. aeruginosa HU and MvaT. Recombinant MvaT was purified as a fusion protein (MAL-MvaT) and shown to specifically bind to the ptxR-ptxS intergenic region. A PAO1 isogenic mutant defective in mvaT, PAOΔmvaT, was constructed and characterized. The lysate of PAOΔmvaT failed to bind to the 150 bp probe. The effect of mvaTon ptxS and ptxR expression was examined using real-time PCR experiments. The expression of ptxS was lower in PAOΔ mvaT than in PAO1, but no difference was detected in ptxR expression. These results suggest that MvaT positively regulates ptxS expression by binding specifically to the ptxS upstream region.