OBJECTIVE We evaluated the genetic and molecular basis of high-level resistance to gentamicin and amikacin in 91 clinical isolates of Enterococcus faecalis and Enterococcus faecium in a university hospital in southern Italy from 1987 to 2003. METHODS Antibiotic susceptibility was evaluated by disc diffusion and microdilution methods. Genotyping was performed by PFGE and dendrogram analysis. Aminoglycoside resistance genes were analysed by multiplex PCR. Aminoglycoside resistance gene transfer was performed by filter mating. RESULTS In our strain collection, 44% of E. faecalis and 52% of E. faecium were high-level-resistant to gentamicin. Fifty-two PFGE profiles were identified for E. faecalis and 15 for E. faecium. Although the majority of PFGE patterns were single isolates, four patterns (two for E. faecalis and two for E. faecium) were isolated each in 8 and 4, and 6 and 4 different patients, respectively. The aac(6')-Ie-aph(2'')-Ia gene was responsible for high-level resistance to gentamicin and amikacin in E. faecalis and E. faecium; the aph(2'')-Id gene responsible for resistance to gentamicin was also isolated in E. faecium; the aph(3')-IIIa and ant(4')-Ia genes responsible for resistance to amikacin were also isolated in E. faecalis and E. faecium. High-level resistance to gentamicin, along with the aac(6')-Ie-aph(2'')-Ia gene, was transferred at a frequency of about 10(-5) to 10(-8) per recipient cell in 14 of 17 E. faecalis and 3 of 4 E. faecium different genotypes. CONCLUSIONS The spread of the aac(6')-Ie-aph(2'')-Ia gene was responsible for high-level resistance to gentamicin and amikacin among enterococci isolated from patients in our geographical area.