Vancomycin-resistant enterococci (VRE) have become a major cause of nosocomial infections. The increase of vancomycin-resistant Enterococcus faecium (VR-Efm) in an intensive care unit (ICU) of an Italian university hospital from 2003 through 2004, led us to evaluate the phenotypic and genetic features of these strains. The prevalence of different bacterial species in this ICU is described. The antibiotic resistance profiles of VR-Efm strains, their van-genotype and pulsed-field gel electrophoresis (PFGE) profiles were also analyzed. From January 2003 to December 2004, VR-Efm strains were collected from several biological samples. Bacteria were identified using standard biochemical reactions and automated systems. Antibiotic susceptibility was evaluated by disk diffusion and microdilution methods. Resistance to glycopeptides was confirmed by the E test. Vancomycin-resistant genotypes (vanA, vanB) were identified by PCR. Strains were typed by PFGE. Fifty E. faecium strains were isolated from a total of 700 patients. Of these, 26 were vancomycin-resistant and were isolated from 26 different patients. We also found one strain with resistance to linezolid. The vanA genotype was identified in 20/26 strains and vanB in the remaining strains. A major pulsed-field cluster ("A") was identified. In this cluster, 14 strains were identified (A1-A14) and 25 out of 26 VR-Efm belonged to it. Only one strain showed a different pattern (strain type "B"). All isolates with the vanA genotype belonged to cluster "A", therefore five out of six isolates with the vanB genotype belonged to cluster A. The only strain with type B pattern was the vanB genotype. Isolation of VR-Efm was very frequent (52%) in our cohort of patients and the vanA genotype was the most frequent (77%). We found 25 out of 26 VR-E. faecium strains to be epidemiologically related by PFGE (cluster A). Strains with distinct genotypes shared closely related PFGE profiles. The occurrence of one major cluster among patients of a single unit indicated intra-facility VRE transmission.