Kanamycin group antibiotics were subjected to enzymatic acetylation by a cell free extract containing an aminoglycoside 3-N-acetyltransferase, AAC(3)-X, derived from Streptomyces griseus SS-1198PR. Characterization of the incubated reaction mixtures by TLC and antibiotic assay revealed that a product retaining activity was specifically formed from arbekacin, an anti-MRSA semisynthetic aminoglycoside. The structural determination demonstrated that acetylation occurred at the 3"-amino group in arbekacin and amikacin, and at the 3-amino group in dibekacin as in the case of kanamycin. These results should reflected the effect of the (S)-4-amino-2-hydroxybutyryl side chain which is present in arbekacin and amikacin, but absent in dibekacin and kanamycin. The 3"-N-acetylation is the first finding in the enzymatic modifications of aminoglycoside antibiotics. 3"-N-Acetylarbekacin showed antibiotic activity as high as that of 2'-N-acetylarbekacin reported previously, whereas 3"-N-acetylamikacin showed no substantial activity. Thus, our results illuminated a novel aspect of arbekacin distinct from the other aminoglycosides.