Objectives: To investigate an outbreak of glycopeptide-resistant Enterococcus faecalis (GREF) in the intensive care unit (ICU) of 'G. Gennimatas' General Hospital, Athens, Greece., Materials and Methods: Between August 2000 and November 2001, 20 highly GREF isolates were recovered from severe infections of separate patients in the ICU. The isolates were tested by PCR, PFGE, mating experiments and plasmid analysis., Results: All isolates carried the vanA gene. Nineteen isolates fitted to one clone by macrorestriction analysis with four subclones being consecutively detected. Each subclone seemed to predominate for a specific time period. Additionally, four GREF isolates related to the ICU clone were recovered from other wards of the hospital., Conclusions: Our findings indicate that a monoclonal GREF outbreak persisted for more than 1 year in a large Greek hospital. The rate of GREF isolation declined after the application of infection control measures.