Despite studies of the pathologic and microbiologic aspects of the lung in cystic fibrosis (CF), there is a lack of information on the lung localization of bacterial pathogens. Bacteriologic data come from cultures of sputum or accessible lung effluent from bronchoscopy. Our objective was to localize Pseudomonas aeruginosa (PA) in situ in order to provide descriptive data on the relationship between the presence of PA and disease of the surrounding tissue. Using stored, uncut blocks of preserved lung, we deparaffinized, cut, mounted, and reacted them with high-titer rabbit sera made to a mucoid strain of PA. The tissues were then reacted with a second antibody, biotinylated goat antirabbit immunoglobulin, developed using a peroxidase technique and counterstained with hematoxylin. PA organisms stained with heavy brown deposits and this was species-specific. In five patients with CF chronically colonized with PA, the organisms could easily be localized in multiple sections. Microscopic study demonstrated that location was generally endobronchiolar and associated with bronchiolar obliterative changes, mainly in small (less than 1 mm) airways. Extraluminal PA organisms were rare even when there was chronic interstitial inflammation. This study demonstrated the presence of PA at the location where physiologic damage in CF is most severe--the small bronchioles--strengthening the association between PA and pulmonary deterioration in CF.