From a rare actinomycete strain #8 isolated from soil as arbekacin (ABK) resistant, we cloned a gene segment (0.9 kb) conferring multiple resistance to aminoglycoside (AG) antibiotics with 6'-NH2 including semisynthetic ones except ABK and neomycin (NM). Enzymatic modification using cell free extracts from Streptomyces lividans TK21/pANT-S2 carrying the cloned gene revealed that the gene coded for an AG 6'-acetyltransferase [AAC(6')] capable of acetylating all of the tested AGs with 6'-NH2 including semisynthetic ones and astromicin. The substrate specificity of the enzyme was thus similar to that of AAC(6')-Ie of Enterococcus faecalis. Antibiotic assay revealed a weak but clear antibiotic activity of 6'-N-acetylABK (8% of ABK activity) in contrast with substantial inactivation by the AAC(6') of the other AGs including amikacin and isepamicin. The NM acetylation by the AAC(6') also did not result in NM inactivation. It seems thus likely that AAC(6')-dependent resistance to ABK and NM, if it emerges, will remain at low level.