Activation of algD by AlgR is essential for mucoidy, a virulence factor expressed by Pseudomonas aeruginosa in cystic fibrosis. Two AlgR-binding sites, RB1 and RB2, located far upstream from the algD mRNA start site, are essential for the high-level activity of algD. However, the removal of RB1 and RB2 does not completely abolish inducibility of algD in response to environmental signals. In this work, a third binding site for AlgR, termed RB3, near the algD mRNA start site was characterized. Deletion of RB3 abrogated both the AlgR-binding ability and the residual inducibility of the algD promoter. DNase I footprinting analysis of RB3 resulted in a protection pattern spanning nucleotides -50 to -30. Eight of 10 residues encompassing a continuous region of protection within RB3 (positions -45 to -36) matched in the inverted orientation the conserved core sequence (ACCGTTCGTC) of RB1 and RB2. Quantitative binding measurements of AlgR association with RB1, RB2, and RB3 indicated that AlgR had significantly lower affinity for RB3 than for RB1 and RB2, with differences in the free energy of binding of 1.05 and 0.93 kcal/mol (4.39 and 3.89 kJ/mmol), respectively. Altering the core of RB2 to match the core of RB3 significantly reduced AlgR binding. Conversely, changing the core of RB3 to perfectly match the core of RB2 (mutant site termed RB3*) improved AlgR binding, approximating the affinity of RB2. RB3*, in the absence of the far upstream sites, showed an increase in activity, approaching the levels observed with the full-size algD promoter. Changing 4 nucleotides in two different combinations within the core of RB3 abolished the binding of AlgR to this site and resulted in a significant reduction of promoter activity in the presence of the far upstream sites. Thus, (i) the core sequence is essential for AlgR binding; (ii) the three binding sites, RB1, RB2, and RB3, are organized as an uneven palindrome with symmetrical sequences separated by 341 and 417 bp; and (iii) all three sites participate in algD activation.