A prospective study was conducted to determine the prevalence of aminoglycoside-resistant Staphylococcus aureus and coagulase-negative staphylococci before and after the introduction of amikacin as the sole aminoglycoside used in our burn unit, adult intensive care unit, and neonatal intensive care unit. Pharyngeal or endotracheal cultures, as well as superficial surveillance cultures, were collected weekly during the following four study periods: all units for 4 months before amikacin introduction, all units 4 to 8 months after, all units 12 to 13 months after, and the neonatal intensive care unit 30 months after. A total of 2,613 strains of coagulase-negative staphylococci and 316 strains of S. aureus were obtained from 916 patients. During the course of the study, amikacin-resistant coagulase-negative staphylococci increased from 0 to 22%, colonizing 43% of patients, whereas no amikacin-resistant S. aureus was detected. During the preamikacin survey, 68% of the coagulase-negative staphylococci and 12% of the S. aureus strains were resistant to tobramycin and gentamicin. This resistance did not decrease after amikacin was introduced. Initially, 83% of the aminoglycoside-resistant coagulase-negative staphylococci were resistant to both tobramycin and gentamicin. During the last surveillance this value dropped to 40%, and 48% of the strains had become resistant to all three aminoglycosides. Resistance to aminoglycosides, including amikacin, develops quickly in coagulase-negative staphylococci from clinical areas where these antimicrobial agents are widely used. However, aminoglycoside resistance in S. aureus is much less frequent.