In Pseudomonas aeruginosa, the molecular regulation of virulence factors and secondary metabolites is tightly controlled. This control involves several signal-mediated regulatory networks, including the GacS-GacA system and quorum sensing. Recently, the posttranscriptional repressor protein RsmA has been implicated in secondary metabolite production. RsmA is postulated to work in tandem with an as yet unidentified regulatory RNA molecule in a manner analogous to its homologues in other bacteria. Here we have identified a gene encoding an untranslated regulatory RNA (RsmB), located in the rpoS/ fdxA intergenic region of the P. aeruginosa PAO1 genome. Overexpression of rsmB in P. aeruginosa resulted in an increase in N-acyl-homoserine lactone, pyocyanin and elastase production compared with a marked decrease when rsmA was overexpressed. Mutation of rsmB resulted in a decrease in AHL production compared to wild type. We propose that RsmB is the cognate regulatory RNA of RsmA in P. aeruginosa. The global regulator GacA was not absolutely required for rsmB transcription in P. aeruginosa, as is the case in Pseudomonas fluorescens. However, GacA influenced the kinetics of rsmB transcription in that in late stationary phase the gacA mutant showed a substantial reduction in rsmB transcript levels compared to wild type. RsmA also influenced rsmB; in an rsmA mutant, the steady state level of rsmB transcript was reduced and this was due to a decrease in the transcription of rsmB. A balance in the levels of RsmA and RsmB may be an autoregulatory mechanism to ensure that RsmA is tightly controlled, as might be expected for such a potent global repressor.