Individuals with cystic fibrosis (CF) are commonly colonized with Pseudomonas aeruginosa. The chronic infections caused by P. aeruginosa are punctuated by acute exacerbations of the lung disease, which lead to significant morbidity and mortality. As regulators of virulence determinants, P. aeruginosa quorum-sensing systems may be active in the chronic lung infections associated with CF. We have examined the levels of autoinducer molecules and transcript accumulation from the bacterial populations found in the lungs of patients with CF. We detected biologically active levels of N-(3-oxododecanoyl)-L-homoserine (3-oxo-C12-HSL) and N-butyryl-L-homoserine lactone (C4-HSL) in sputum from CF patients. Interestingly, it appears that C4-HSL is less frequently detected than 3-oxo-C12-HSL in the lungs of patients with CF. We also examined the transcription of the autoinducer synthase gene lasI and showed that it is frequently expressed in the lungs of patients with CF. We observed a significant correlation between the expression of lasI and four target genes of the Las quorum-sensing system. Taken together, our results indicate that quorum-sensing systems are active and may control virulence factor expression in the lungs of patients with CF.