Fourteen different aminoglycoside antibiotics (AGs) were challenged with aminoglycoside acetyltransferases (AACs) of actinomycete origin in order to examine their 'double stage activity' that is arbitrarily defined as antibiotic activity retainable after enzymatic modification. In kanamycin (KM)-group AGs tested [KM, dibekacin (DKB), amikacin and arbekacin (ABK)], ABK retained activity after acetylations by AAC(3), AAC(2') and AAC(6'). DKB also retained a weak activity after acetylation by AAC(2'). In gentamicin (GM)-group AGs tested [GM, micronomicin, sisomicin (SISO), netilmicin (NTL) and isepamicin], GM, SISO and NTL retained activites after acetylation by AAC(2'). In neomycin (NM)-group AGs tested [ribostamycin, NM, paromomycin], NM retained activity after acetylation by AAC(6') and AAC(2'). None of astromicin (ASTM)-group AGs tested (ASTM and istamycm B) retained activity after acetylation by AAC(2') and AAC(6'). The activities of acetylated ABK derivatives by AAC(3) and AAC(2') were distinctively high> compared to the others. Streptomyces lividans TK21 containing the cloned aac genes were markedly sensitive to AGs that retained activities after acetylation, indicating the substantial effect of 'double stage activity'.