Transcriptional profiling of Pseudomonas aeruginosa grown under steady-state hyperosmotic stress conditions showed an up-regulation of genes associated with osmoprotectant synthesis, putative hydrophilins, and the type III secretion system with associated cytotoxins. A large number of regulatory genes, including several two-component systems not previously known to be influenced by osmolarity, were differentially expressed by P. aeruginosa in immediate response to hyperosmotic shock.