We have previously shown that the Pseudomonas aeruginosa toxA regulatory protein PtxS autoregulates its own synthesis by binding to a 52-bp fragment. The 3' end of the 52-bp fragment is located 58 bp 5' of the ptxS translation start site. We have identified a 14-bp palindromic sequence (TGAAACCGGTTTCA) within the 52-bp fragment. In this study, we used site-directed mutagenesis and promoter fusion experiments to determine if PtxS binds specifically to this palindromic sequence and regulates ptxS expression. We have also tried to determine the roles of specific nucleotides within the palindromic sequence in PtxS binding and ptxS expression. Initial promoter fusion experiments confirmed that the 52-bp fragment does not overlap with the region that carries the ptxS promoter activity. PtxS binding was eliminated upon the deletion of the 14-bp palindromic sequence from the 52-bp fragment. In addition, the deletion of the 14-bp sequence caused a significant enhancement in ptxS expression in the P. aeruginosa strain PAO1 and the ptxS isogenic mutant PAO::ptxS. Mutation of specific nucleotides within the 14-bp sequence eliminated, reduced, or had no effect on PtxS binding. However, mutations of several of these nucleotides produced a significant increase in ptxS expression in both PAO1 and PAO::ptxS. These results suggest that (i) the 14-bp palindromic sequence and specific nucleotides within it play a role in PtxS binding and (ii) deletion of the palindromic sequence or changing of certain nucleotides within it interferes with another mechanism that may regulate ptxS expression.