A total of 55 Enterococcus faecalis and 21 Enterococcus faecium non-replicate isolates were obtained from routine clinical specimens, during a 1 year period, in a tertiary care hospital in Athens, Greece. The most common isolation site was the urinary tract (44% of E. faecalis and 33% of E. faecium isolates). No vancomycin resistance was detected. Ampicillin-resistant isolates did not produce beta-lactamase. High-level gentamicin resistance was detected in 22% and 0% of E. faecalis and E. faecium isolates, respectively. The corresponding figures for high-level streptomycin resistance were 40% and 33%. The aminoglycoside-modifying enzyme gene aac(6')+aph(2") was detected by PCR in 10 of 12 high-level gentamicin-resistant E. faecalis isolates, and the ant(6)-I gene in all high-level streptomycin-resistant isolates of both species. DNA fingerprinting by PFGE grouped 31 of 55 E. faecalis isolates into 10 clusters, and 10 of 21 E. faecium isolates into two clusters, containing two to seven isolates each. Two E. faecalis PFGE types, comprising isolates expressing high-level aminoglycoside resistance, and not observed among non-high-level aminoglycoside-resistant strains, were disseminated in building A of the hospital. In contrast, high-level aminoglycoside resistance seemed to have been acquired nosocomially by a number of genotypically different E. faecium types. Molecular typing was therefore instrumental in understanding the differences in the mode of spread and acquisition of high-level aminoglycoside resistance among these two different enterococcal species.