Between April 1992 and December 1993, 80 Xanthomonas maltophilia isolates were collected from 63 patients in three acute-care hospitals in Calgary, Alberta, Canada. On the basis of Centers for Disease Control and Prevention definitions, 48 patients had nosocomial and 15 had community-acquired X. maltophilia. Thirty-eight of the patients were colonized and 25 were infected. Sixty-four percent of patients who acquired X. maltophilia in the intensive care unit (ICU) became infected, whereas 32% of patients in a non-ICU setting became infected. ICU patients tended to be hospitalized for a shorter period of time than non-ICU patients before the onset of X. maltophilia infection. Regardless of being colonized or infected, all patients had debilitating conditions, with respiratory disease being the most common underlying illness (35%). Forty-two patients (88%) with hospital-acquired X. maltophilia received prior antibiotic therapy which included gentamicin, tobramycin, ceftazidime, piperacillin, and imipenem. Agar dilution MICs showed that patient isolates were resistant to these antimicrobial agents that patients had received. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA revealed that six epidemiologically linked patient isolates from the ICU of one acute-care hospital had identical DNA profiles. In contrast, isolates from patients from the other two hospitals had unique genotype profiles (n = 57) regardless of the presence or absence of an epidemiologic association. In these patients there was genetic evidence against the acquisition of a resident hospital clone. These results indicate that pulsed-field gel electrophoresis can resolve genotypically distinct strains of X. maltophilia and, consequently, is a useful tool for evaluating nosocomial infections caused by X. maltophilia.