Stenotrophomonas maltophilia was isolated from the respiratory tracts of 41 (25%) of 163 children attending our pediatric cystic fibrosis unit between September 1993 and December 1995. The extents of S. maltophilia contamination of environmental sites frequented by these patients were investigated with a selective medium incorporating vancomycin, imipenem, and amphotericin B. Eighty-two isolates of S. maltophilia were cultured from 67 different environmental sites sampled between January and July 1996. The organism was widespread in the home environment, with 20 (36%) and 25 (42%) of sampled sites positive in the homes of colonized and noncolonized patients, respectively. In the nosocomial setting, it was isolated from 18 (32%) sites in the hospital ward and from 4 (17%) sites in the outpatient clinic area. The most common sites of contamination were sink drains, faucets, and other items frequently in contact with water. All environmental and clinical isolates were genotyped with enterobacterial repetitive intergenic consensus sequences as primers. A total of 33 of the 41 patients were colonized with unique strains, and four pairs of patients shared strains. Further characterization by pulsed-field gel electrophoresis after digestion with XbaI found that there was no evidence of patient-to-patient transmission; however, there was some evidence that a small number of patients may have acquired the organism from the hospital environment. Resampling of environmental sites in the hospital ward in January 1997 revealed evidence of genetic drift, complicating the accurate determination of environmental sources for clinical strains. The source of the majority of S. maltophilia strains colonizing the respiratory tracts of these patients with cystic fibrosis remained uncertain but may have represented multiple, independent acquisitions from a variety of environmental sites both within and outside the hospital.