Aminoglycoside modifying enzymes (AMEs) are major factors which confer aminoglycoside resistance on bacteria. Distribution of genes encoding seven AMEs was investigated by multiplex PCR for 279 recent clinical isolates of enterococci derived from a university hospital in Japan. The aac(6′)-aph(2″), which is related to high level gentamicin resistance, was detected at higher frequency in Enterococcus faecalis (42·5 %) than in Enterococcus faecium (4·3 %). Almost half of E. faecalis and E. faecium isolates possessed ant(6)-Ia and aph(3′)-IIIa. The profile of AME gene(s) detected most frequently in individual strains of E. faecalis was aac(6′)-aph(2″)+ant(6)-Ia+aph(3′)-IIIa, and isolates with this profile showed high level resistance to both gentamicin and streptomycin. In contrast, AME gene profiles of aac(6′)-Ii+ant(6)-Ia+aph(3′)-IIIa, followed by aac(6′)-Ii alone, were predominant in E. faecium. Only one AME gene profile of ant(6)-Ia+aph(3′)-IIIa was found in Enterococcus avium. The ant(4′)-Ia and ant(9)-Ia, which have been known to be distributed mostly among Staphylococcus aureus strains, were detected in a few enterococcal strains. An AME gene aph(2″)-Ic was not detected in any isolates of the three enterococcal species. These findings indicated a variety of distribution profiles of AME genes among enterococci in our study site.