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Isolates of Staphylococcus aureus obtained from a Brazilian university hospital were characterized in relation to resistance to gentamicin and related aminoglycosides. Thirty-six isolates were susceptible to methicillin (MSSA) and 14 were resistant (MRSA). All isolates were sensitive to nucleic acid-binding compounds. All MRSA isolates and one MSSA isolate were demonstrated to be resistant to gentamicin and were coincidentally resistant to amikacin, kanamycin, neomycin and tobramycin. Among the gentamicin sensitive MSSA isolates, five isolates were found to be resistant only to kanamycin/neomycin. The resistance to gentamicin (and related aminoglycosides: kanamycin and tobramycin) must be due to AAC(6′)-APH(2″) activity. As these isolates also showed resistance to neomycin, they must carry an additional genetic element, probably the one responsible for APH(3′)III activity, which accounts for the high level of resistance to kanamycin and to amikacin. The resistance to kanamycin/neomycin in the gentamicin sensitive isolates could not be attributed to the AAD(4′)(4″) activity because of the tobramycin sensitivity, and so could be ascribed to the APH(3′)III activity. Curing and transfer experiments, as well as electrophoresis procedures, indicate that gentamicin resistance in Staph. aureus strains here studied has, characteristically, chromosomal localization.