Pseudomonas aeruginosa controls the secretion of extracellular virulence factors, including rhamnolipids and LasB elastase, by the las and rhl quorum-sensing systems. Here, we mutated the dksA gene of P. aeruginosa by insertion of an Omega-Hg cassette. The mutant displayed growth rates similar to that of the wild type in rich medium but was impaired in growth in defined minimal medium. Production of rhamnolipids and LasB elastase by the dksA mutant was only 4 and 10%, respectively, of wild-type levels. These defects could be partially complemented by introduction of the plasmid-encoded dksA genes from P. aeruginosa or Escherichia coli. In the dksA mutant, the expression of rhlI was increased early during exponential growth, but expression of other quorum-sensing regulator genes-lasR, lasI, and rhlR-was not affected. Although the transcription of the lasB and rhlAB genes was comparable between the dksA mutant and the wild-type strain in peptone tryptic soy broth medium, we observed reduced translation of both genes in the dksA mutant. Similarly, we found that full translation of lasB and rhlAB genes in E. coli also requires the dksA gene. DksA is therefore a novel regulator involved in the posttranscriptional control of extracellular virulence factor production in P. aeruginosa.