Quorum sensing systems are used by a number of Gram-negative bacterial species to regulate specific sets of genes in a cell density-dependent manner. Quorum sensing involves synthesis and detection of extracellular signals termed autoinducers. As shown in recombinant Escherichia coli, the Pseudomonas aeruginosa autoinducer (PAI) N-(3-oxododecanoyl)homoserine lactone, together with the lasR gene product, activate the P. aeruginosa lasB gene. In this study, PAI was shown to activate lasB-lacZ expression in a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of the lac promoter. The concentration of PAI necessary for half-maximal activation of the lasB-lacZ fusion was approximately 1 microM, which is within the range of PAI levels found in P. aeruginosa culture fluids. The effect of PAI on a P. aeruginosa lasR mutant containing a plasmid with lasR under the control of its own promoter and containing the lasB-lacZ fusion was also tested. Although extracts of culture fluid activated the lasB promoter in this construct, concentrations of PAI as high as 10 microM did not. This indicates the presence of a second extracellular factor (factor 2) that is required for lasB activation in P. aeruginosa when lasR is controlled by its own promoter but not when lasR is controlled by a strong foreign promoter. Factor 2 was shown to be N-butyrylhomoserine lactone. Although recombinant E. coli cells containing the PAI synthase gene, lasI, produce PAI, these cells do not produce factor 2. Furthermore, a P. aeruginosa mutant that produced about 0.1% of the wild-type level of PAI made about 5% of the wild-type level of factor 2. This indicates that factor 2 synthesis results from the activity of a gene product other than PAI synthase. The role of factor 2 in virulence gene regulation remains to be determined, but this compound may affect the expression of lasR, which in turn activates transcription of numerous virulence genes in the presence of sufficient PAI. Apparently, multiple quorum sensing systems can occur and interact with each other in a single bacterial species.