AmpD indirectly regulates the production of AmpC beta-lactamase via the cell wall recycling pathway. Recent publications have demonstrated the presence of multiple ampD genes in Pseudomonas aeruginosa and Escherichia coli. In the prototype P. aeruginosa strain, PAO1, the three ampD genes (ampD, ampDh2, and ampDh3) contribute to a stepwise regulation of ampC beta-lactamase and help explain the partial versus full derepression of ampC. In the present study, the roles of the three ampD homologs in nine clinical P. aeruginosa isolates with either partial or full derepression of ampC were evaluated. In eight of nine isolates, decreased RNA expression of the ampD genes was not associated with an increase in ampC expression. Sequence analyses revealed that every derepressed isolate carried mutations in ampD, and in two fully derepressed strains, only ampD was mutated. Furthermore, every ampDh2 gene was of the wild type, and in some fully derepressed isolates, ampDh3 was also of the wild type. Mutations in ampD and ampDh3 were tested for their effect on function by using a plasmid model system, and the observed mutations resulted in nonfunctional AmpD proteins. Therefore, although the sequential deletion of the ampD homologs of P. aeruginosa can explain partial and full derepression in PAO1, the same model does not explain the overproduction of AmpC observed in these clinical isolates. Overall, the findings of the present study indicate that there is still an unknown factor(s) that contributes to ampC regulation in P. aeruginosa.