Pseudomonas gladioli was isolated from 11 patients with cystic fibrosis. It resembled Pseudomonas cepacia on the selective and differential medium OFPBL, producing yellow colonies after 48 to 72 h of incubation. Isolates were characterized biochemically, by DNA hybridization, and by cellular fatty acid analysis. A review of the clinical status of selected patients colonized by P. gladioli did not reveal any apparent association of this organism with infectious complications of cystic fibrosis. Thus, the clinical implications may differ depending on which of these two closely related species is reported by laboratories. Determination of the fatty acid profile of isolates by gas chromatography may be a useful adjunct to biochemical characterization as a means of identification. In contrast to P. cepacia, most isolates of P. gladioli contained 3-OH C10:0 fatty acid under the growth conditions used.