Biosynthesis of biosurfactant rhamnolipids by Pseudomonas aeruginosa depends on two hierarchical quorum sensing systems, LasRI and RhlRI, which synthesize and sense the signal molecules N-(3-oxododecanoyl)-L-homoserine lactone (3OC(12)-HSL) and N-butyryl-L-homoserine lactone (C(4)-HSL), respectively. The Pseudomonas Quinolone Signal (PQS) is a third cell-to-cell signal molecule connecting these two systems, and its precursor, 2-heptyl-4-quinolone (HHQ), also constitutes a signal. The chronology of the production of signal molecules and rhamnolipids was determined during growth in PPGAS medium. Hyperosmotic condition (0.5 M NaCl) moderately affected growth, and led to intra-cellular accumulation of compatible solutes. Production of signal molecules was delayed and their highest concentrations were 2.5 to 5 fold lower than in NaCl-free PPGAS, except for HHQ, the highest concentration of which was increased. The presence of NaCl prevented rhamnolipid synthesis. When the osmoprotectant glycine betaine was added to PPGAS/NaCl medium, it was imported by the cells without being metabolized. This did not improve growth, but reestablished the time-courses of HSL and HHQ accumulation and fully or partially restored the HSL and PQS levels. It also partially restored rhamnolipid production. Quantification of mRNAs encoding enzymes involved in HSL, PQS, and rhamnolipid biosyntheses confirmed the effect of hyperosmotic stress and glycine betaine at the gene expression level.