Transcriptional activation of the Pseudomonas aeruginosa algD gene results in high-level synthesis of the capsular polysaccharide alginate, an important P. aeruginosa virulence factor expressed in cystic fibrosis (CF) patients with chronic pulmonary disease. In this study, electrophoretic mobility-shift assays were used to identify a novel protein (AlgZ), which binds specifically to a sequence located 280 bp upstream of the algD promoter. While AlgZ-binding activity did not require the response regulators AlgB or AlgR, expression of AlgZ was found to be absolutely dependent on the alternative sigma factor AlgT. Electrophoretic mobility-shift assays and copper-phenanthroline foot-printing localized AlgZ binding to a 36 bp algD region, which includes several helical repeats. A collection of alginate-producing (mucoid) and non-mucoid P. aeruginosa strains, derived from CF patients, was characterized for AlgZ-binding activity. In all cases, AlgZ binding to algD sequences was observed when extracts derived from mucoid P. aeruginosa CF isolates were examined. However, this binding activity was not present when extracts from non-mucoid P. aeruginosa CF isolates were tested. Oligonucleotide mutagenesis was employed to create an algD allele with a 4 bp mutation in the predicted AlgZ-binding site (algD38) and a heterologous substitution allele (algD40), in which the entire AlgZ-binding site was replaced with a nonspecific DNA sequence of identical size. When the algD38 mutation was cloned into an algD-cat transcriptional fusion, this resulted in a 28-fold reduction in algD expression, whereas the algD40 mutation abolished algD transcription, indicating that AlgZ acts as an activator of algD transcription. These results support the hypothesis that activation of algD involves the formation of a high-order looped structure allowing for multivalent contacts between AlgZ, AlgR and RNA polymerase containing the alternative sigma factor AlgT. Characterization of the molecular details of algD activation will provide insights into the control of other prokaryotic and eukaryotic promoters that utilize multiple activators.