We analyzed for the presence of the aacA/aphD gene in arbekacin resistant-Staphylococcus aureus. Tested strains were 50 clinically isolated Staphylococcus aureus that had an MIC of more than 2 micrograms/ml for arbekacin. Among the primary cultures, aacA/aphD genes were detected in 42 of 50 strains(84%) overall, including 34 of 41 Methicillin resistant Staphylococcus aureus(MRSA) strains, and 8 of 9 strains of Methicillin sensitive Staphylococcus aureus(MSSA). After 10 or 20 passages, the aacA/aphD gene was lost in 5 strains of MRSA and 2 strains of MSSA. In the strains that lost the gene, the MIC of arbekacin was reduced remarkably in 3 strains of MRSA and 2 strains of MSSA. Further, MICs of arbekacin decreased along with passages even in the strains that did not loose the aacA/aphD gene, as well as in the strains that did not possess the aacA/aphD gene originally. The fact that MIC values are easily reduced with in vitro passage is important to consider when performing the arbekacin-resistance test for Staphylococcus aureus.