Direct electron microscopic examination of postmortem lung material from cystic fibrosis patients infected with Pseudomonas aeruginosa has shown that these bacterial cells form distinct fiber-enclosed microcolonies in the infected alveoli. Similar examination of bronchoscopy material from infected cystic fibrosis patients showed that the fibres of the enveloping matrix are definitely associated with the bacterial cells. The fibers of the extracellular matrix stain with ruthenium red and are therefore presumed to be polyanionic. When mucoid strains of P. aeruginosa were recovered from cystic fibrosis patients and grown in a suitable liquid medium, they were found to produce large microcolonies whose component cells were embedded in a very extensive matrix of polyanionic fibers that could be stabilized by reaction with antibodies to prevent collapse during the dehydration steps of preparation for electron microscopy. When these mucoid strains of P. aeruginosa were used to produce pulmonary infections of rats by the agar bead method, the infected alveoli contained large fiber-enclosed bacterial microcolonies. We conclude that the cells of P. aeruginosa that infect cystic fibrosis patients form microcolonies that are enveloped in a fibrous anionic matrix and that these microcolonies can be duplicated in in vitro cultures and in animal model systems.