Enterococcus casseliflavus UC73 is a clinical blood isolate with high-level resistance to gentamicin. DNA preparations from UC73 failed to hybridize with intragenic probes for aac(6')-Ie-aph(2'')-Ia and aph(2'')-Ic. A 4-kb fragment from UC73 was cloned and found to confer resistance to gentamicin in Escherichia coli DH5alpha transformants. Nucleotide sequence analysis revealed the presence of a 906-bp open reading frame whose deduced amino acid sequence had a region with homology to the aminoglycoside-modifying enzyme APH(2'')-Ic and to the C-terminal domain of the bifunctional enzyme AAC(6')-APH(2''). The gene is designated aph(2'')-Id, and its observed phosphotransferase activity is designated APH(2'')-Id. A PCR-generated intragenic probe hybridized to the genomic DNA from 17 of 118 enterococcal clinical isolates (108 with high-level gentamicin resistance) from five hospitals. All 17 were vancomycin-resistant Enterococcus faecium isolates, and pulsed-field typing revealed three distinct clones. The combination of ampicillin plus either amikacin or neomycin exhibited synergistic killing against E. casseliflavus UC73. Screening and interpretation of high-level aminoglycoside resistance in enterococci may need to be modified to include detection of APH(2'')-Id.