The destructive pulmonary inflammation associated with Pseudomonas aeruginosa colonization is caused, in part, by the production of the chemokine IL-8, which recruits neutrophils into the lung. The Pseudomonas autoinducer, N-3-oxododecanoyl homoserine lactone (3-O-C12-HSL), is a small lipid-soluble molecule that is essential in the regulation of many P. aeruginosa virulence factors, but little is known about how it affects eukaryotic cells. In this report we demonstrate that 3-O-C12-HSL is a potent stimulator of both IL-8 mRNA and protein from human fibroblasts and epithelial cells in vitro. The IL-8 produced from these 3-O-C12-HSL-stimulated cells was found to be functionally active by inducing the chemotaxis of neutrophils. To determine a mechanism for this IL-8 induction, deletion constructs of the IL-8 promoter were examined. It was found that the DNA region between nucleotides -1481 and -546 and the transcription factor NF-kappaB were essential for the maximal induction of IL-8 by 3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a shift with both AP-2 and NF-kappaB consensus DNA. The activation of NF-kappaB and subsequent production of IL-8 were found to be regulated by a mitogen-activated protein kinase pathway. These findings support the concept that the severe lung damage that accompanies P. aeruginosa infections is caused by an exuberant neutrophil response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the mechanisms of 3-O-C12-HSL activation of lung structural cells may provide a means to help control lung damage during infections with P. aeruginosa.