Using a polyphasic approach (including cellular protein and fatty acid analysis, biochemical characterization, 16S ribosomal DNA sequencing, and DNA-DNA hybridizations), we characterized 51 bacterial isolates recovered from respiratory secretions of cystic fibrosis (CF) patients. Our analyses showed that 24 isolates belong to taxa that have so far not (or only rarely) been reported from CF patients. These taxa include Acinetobacter sp., Bordetella hinzii, Burkholderia fungorum, Comamonas testosteroni, Chryseobacterium sp., Herbaspirillum sp., Moraxella osloensis, Pandoraea genomospecies 4, Ralstonia gilardii, Ralstonia mannitolilytica, Rhizobium radiobacter, and Xanthomonas sp. In addition, one isolate most likely represents a novel Ralstonia species, whereas nine isolates belong to novel taxa within the ␣-Proteobacteria. Eight of these latter isolates are classified into the novel genus Inquilinus gen. nov. as Inquilinus limosus gen. nov., sp. nov., or as Inquilinus sp. The remaining 17 isolates are characterized as members of the family Enterobacteriaceae. The recovery of these species suggests that the CF lung is an ecological niche capable of supporting the growth of a wide variety of bacteria rarely seen in clinical samples. Elucidation of the factors that account for the association between these unusual species and the respiratory tract of CF patients may provide important insights into the pathophysiology of CF infection. Because accurate identification of these organisms in the clinical microbiology laboratory may be problematic, the present study highlights the utility of reference laboratories capable of identifying unusual species recovered from CF sputum.Cystic fibrosis (CF), an autosomal-recessive hereditary disease caused by mutations in the CF transmembrane conductance regulator gene, is characterized by the production of abnormally thickened, viscous mucus and a disturbance in electrolyte transport across epithelial membranes (34). Defects in the CF transmembrane conductance regulator gene mainly affect the respiratory tract and the pancreas, and exacerbations of pulmonary infections cause significant morbidity and mortality in persons with CF (16). Typical CF pathogens include Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, and Burkholderia cepacia complex organisms (16), but other glucose nonfermenters such as Ralstonia pickettii, Alcaligenes xylosoxidans, Stenotrophomonas maltophilia, and Burkholderia gladioli can be recovered from CF sputum culture as well (3,16). Correct identification of CF pathogens is important, since it underlies effective infection control measures and therapuetic intervention (24,25,36); however, several studies have shown that the identification of CF pathogens, including B. cepacia complex organisms, is far from straightforward (3,8,22,28).Here we report the characterization of 51 bacterial isolates recovered from respiratory secretions of CF patients. The majority of these isolates were identified by referring microbiology laboratories as B. ...