The expression of the bifunctional aminoglycoside inactivating enzyme 6'-N-aminoglycoside acetyltransferase-2"-O-aminoglycoside phosphotransferase is the most important mechanism of high-level aminoglycoside resistance in Staphylococcus and Enterococcus. The enzyme is unique because it presents two different aminoglycoside-modifying activities located in different regions of the molecule. The gene aac(6')-aph(2") which encodes the synthesis of the enzyme is present in Tn4100-like transposons which are inserted both in R plasmids and the chromosomes of aminoglycoside-resistant isolates. The genetic structure of aac(6')-aph(2")-containing isolates indicates that their origin is not clonal, but plasmid conjugation together with multiple insertion events are in the basis of the rapid spread of aminoglycoside resistance among Gram-positive bacteria. There is not any prevalent genetic linkage of aac(6')-aph(2") with other antibiotic-resistance determinant. However, most methicillin resistant Staphylococcus strains present also high-level aminoglycoside resistance as the consequence of constant antibiotic pressure. This situation could change in the next future with the reported reemergence of gentamicin-susceptible MRSA isolates. Recent data show that inhibitors of eukaryotic protein kinases inhibit as well the aminoglycoside phosphotransferase activity. This effect indicates a common structure for these two families of proteins and opens the possibility for a meaningful survey of inhibitors of 6'-N-aminoglycoside acetyltransferase-2"-O-aminoglycoside phosphotransferase useful in clinical practice.