To investigate the dissemination of vancomycin-resistant Enterococcus faecium (VREF) in a 728-bed tertiary-care hospital, all clinical VREF isolates recovered from June 1992 to June 1997 were typed by pulsed-field gel electrophoresis, and the transfer histories of the patients were documented. A total of 413 VREF isolates from urine (52%), wounds (16%), blood (11%), catheter tips (6%), and other sites (15%) were studied. VREF specimens mostly came from patients on wards (66%) but 34% came from patients in an intensive care unit. The number of VREF isolates progressively increased over time, with higher rates of isolation during the winter months and lower rates in the late summer months. Four distinct banding patterns were detected by pulsed-field gel electrophoresis among 316 samples (76%). Strain A (122 samples; 30%) appeared in June 1992 as the first VREF strain and was found until December 1994 throughout the entire hospital. Type B (92 samples; 22%) was initially detected in January 1994 and disappeared in November 1996. Strain C (10 samples; 2%) was limited to late 1996 and early 1997. Strain D (92 samples; 22%) showed two major peaks during March 1996 to August 1996 and January 1997 to February 1997. Unrelated strains (97 samples; 24%) appeared 1 year after the appearance of the first VREF isolate, and the numbers increased slightly over the years. Nosocomial acquisition (i.e., no known detection prior to admission and first isolation from cultures performed with samples retrieved 2 days after hospitalization) was found for 316 (91%) of 347 patients. Despite the implementation of Centers for Disease Control and Prevention guidelines, the proportion of related strains and high number of nosocomial cases of infection indicate a high transmission rate inside the hospital. The results imply an urgent need for stringent enforcement of more effective infection control measures.