A multiple-primer PCR was used to identify genes encoding aminoglycoside-modifying enzymes in 381 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The technique used three sets of primers delineating specific DNA fragments of the aph(3)-III, ant(4)-I, and aac(6)-aph(2؆) genes, which influence the MICs of gentamicin, tobramycin, and lividomycin. Isolates with none of the three genes detected were susceptible to all three agents. Isolates with the aph(3)-III gene showed resistance to lividomycin (MIC > 1,024 g/ml), and those with the ant(4)-I gene were resistant to tobramycin (MIC > 8 g/ml). Isolates with only the aac(6)-aph(2؆) gene were resistant to gentamicin (MIC > 8 g/ml) and tobramycin in decreasing order; those with both the ant(4)-I and aac(6)-aph(2؆) genes also were resistant to gentamicin and tobramycin, but in increasing order. Susceptibility testing, then, could detect specific genes. In 381 Japanese MRSA isolates, the ant (4)