The kinetic constants of the aminoglycoside-modifying enzyme 6'-N-aminoglycoside acetyltransferase (AAC(6')IV) from the clinical strain Staphylococcus epidermidis RYC 13036 differed depending on whether tobramycin and amikacin (glycosamine group) or gentamicin and netilmicin (garosamine group) were used as substrates. Acetylation of the glucosamine antibiotics was highly susceptible to substrate inhibition which increased with pH whereas the garosamine group compounds showed limited substrate inhibition over a wide pH range. These differences in activity correlated with MIC values of S. epidermidis RYC 13036 for different aminoglycosides. Aminosugars moiety and pH markedly influenced the AAC(6')IV-aminoglycoside interactions.