A novel aminoglycoside resistance gene, aac(6′)-Iad, encoding aminoglycoside 6′-N-acetyltransferase, was identified in Acinetobacter genospecies 3 strain A.51. The gene encoded a 144-amino-acid protein, which shared modest identity (up to 36.7%) with some of the aminoglycoside 6′-N-acetyltransferases. The results of high-pressure liquid chromatography assays confirmed that the protein is a functional aminoglycoside 6′-N-acetyltransferase. The enzyme conferred resistance to amikacin, tobramycin, sisomicin, and isepamicin but not to gentamicin. The prevalence of this gene among Acinetobacter clinical isolates in Japan was then investigated. Of 264 Acinetobacter sp. strains isolated from geographically diverse areas in Japan in 2002, 16 were not susceptible to amikacin, and aac(6′)-Iad was detected in 7. Five of the producers of aminoglycoside 6′ -N-acetyltransferase type Iad were identified as Acinetobacter baumannii, and two were identified as Acinetobacter genospecies 3. These results suggest that aac(6′)-Iad plays a substantial role in amikacin resistance among Acinetobacter spp. in Japan.