A total of 36 vancomycin-resistant Enterococcus faecium isolates obtained from 30 patients during a 28-month period in a paediatric university hospital was analysed by pulsed-field gel electrophoresis (PFGE) combined with Southern hybridisation of a vanA-specific DNA probe. All the isolates hybridised with the vanA probe. Seventeen different PFGE patterns and 11 PFGE subtypes were identified among the 36 clinical isolates, and the size of probe-positive bands ranged from c. 30 to 300 kb. These data are consistent with an increase in the overall genomic diversity of vancomycin-resistant E. faecium isolates during the study period. Two periods were distinguished. The prevalence of a single clone in the initial period suggested transmission between patients in three wards. During the following period, multiple genotypes of vancomycin-resistant E. faecium were identified, indicative of multiple introductions or the dissemination of resistance genes by recombinant transposition.