The stringent response is a mechanism by which bacteria adapt to nutritional deficiencies through the production of the guanine nucleotides ppGpp and pppGpp, produced by the RelA enzyme. We investigated the role of the relA gene in the ability of an extracellular pathogen, Pseudomonas aeruginosa, to cause infection. Strains lacking the relA gene were created from the prototypical laboratory strain PAO1 as well as the mucoid cystic fibrosis isolate 6106, which lacks functional quorum-sensing systems. The absence of relA abolished the production of ppGpp and pppGpp under conditions of amino acid starvation. We found that strains lacking relA exhibited reduced virulence in a D. melanogaster feeding assay. In conditions of low magnesium, the relA gene enhanced production of the cell-cell signal N-[3-oxododecanoyl]-l-homoserine lactone, whereas relA reduced the production of the 2-heptyl-3-hydroxy-4-quinolone signal during serine hydroxamate induction of the stringent response. In the relA mutant, alterations in the Pseudomonas quinolone system pathways seemed to increase the production of pyocyanin and decrease the production of elastase. Deletion of relA also resulted in reduced levels of the RpoS sigma factor. These results suggest that adjustment of cellular ppGpp and pppGpp levels could be an important regulatory mechanism in P. aeruginosa adaptation in pathogenic relationships.