Chronic Pseudomonas aeruginosa infections lead to progressive lung tissue destruction in cystic fibrosis (CF) patients. Two bacterial cell-to-cell signals, 3-oxo-C(12)-HSL and C(4)-HSL are required for the production of several extracellular virulence factors. 3-oxo-C(12)-HSL is also required for the development of a differentiated biofilm, induces IL-8 production by epithelial cells and possesses immunomodulatory activities. These two signalling molecules are therefore believed to play a role in the pathogenesis of P. aeruginosa infections, but have never been isolated from infected human tissues. We extracted and quantified the two P. aeruginosa cell-to-cell signals from lung tissues of two CF patients infected by P. aeruginosa. 3-oxo-C(12)-HSL and C(4)-HSL were detected in the lung tissues in fmol/gram, respectively pmol/gram concentrations; the ratio C(4)-HSL/3-oxo-C(12)-HSL exceeded 100 in all tissue samples. Random Amplified Polymorphism DNA genotyping revealed that one genotype was present per lung. In vitro the P. aeruginosa isolates from the two lungs produced 3-oxo-C(12)-HSL, whereas some isolates did not produce detectable C(4)-HSL. Our results suggest that both P. aeruginosa cell-to-cell signals were produced in the lung tissue of these two cystic fibrosis patients.