Background: Chronic rhinosinusitis (CRS) that is refractory to medical or surgical intervention may involve a particularly resistant form of infection known as a bacterial biofilm. Bacterial biofilms are three-dimensional aggregates of bacteria that often are recalcitrant to antibiotics secondary to physical barrier characteristics. To date, all studies investigating biofilms in CRS have been descriptive in either human or animal tissue. To better understand the interactions of bacterial biofilms with respiratory epithelium, we describe an in vitro model of biofilm sinusitis by establishing mature biofilms on airway epithelial air-liquid interface cultures. Methods: Airway epithelial cell cultures were grown on collagen-coated semipermeable support membranes as an air-liquid interface on tissue culture inserts. Confluent air-liquid interface cultures were inoculated with the biofilm-forming PAO-1 strain of Pseudomonas aeruginosa and compared with cultures inoculated with two mutant strains (sad-31 and sad-36) unable to form biofilms. Inoculated tissue transwells were incubated for 20 hours, allowing for biofilm growth. The semipermeable membranes were then harvested and imaged with confocal laser scanning microscopy and scanning electron microscopy. Results: Microscopic analysis revealed the formation of biofilm-forming towers in the PAO-1 inoculated wells. The bacterial biofilms were supported by a viable airway epithelial cell surface monolayer. Conclusion: This study shows a reliable method for analysis of in vitro interactions of bacterial biofilms and airway epithelium. The experimental manipulation of this air-liquid interface model will help explore novel treatment approaches for bacterial biofilm-associated CRS. Copyright © 2008, OceanSide Publications, Inc., U.S.A.