The sequence of seven aac(6')-I genes encoding aminoglycoside 6'-N-acetyltransferases from proteolytic Acinetobacter strains including genomic species 14, 15, 16, and 17 and from ungrouped proteolytic strains 631, 640, and BM2722 was determined. Pulsed-field gel electrophoresis of genomic DNA of these strains and of Acinetobacter sp. 6 CIP A165 digested with SfiI followed by hybridization with rRNA and aac(6')-I specific probes indicated that these genes were located in the chromosome. Phylogenetic analysis of the genes indicated that aac(6')-I of A. baumannii, Acinetobacter ungrouped strain 631, and Acinetobacter sp. 16 formed a cluster (91.5 to 92.3% identity) whereas aac(6')-I of Acinetobacter sp. 15, sp. 17, and Acinetobacter ungrouped strain BM2722 formed another cluster (90.7 to 94.6% identity). A third cluster was constituted by A. haemolyticus and Acinetobacter sp. 6 (83.6% identity). The phylogeny drawn from aac(6')-I sequences was consistent with that based on DNA-DNA hybridization and phenotype comparison. The aac(6')-I genes were all species specific except for aac(6')-Ih located in a 13.7-kb non conjugative plasmid from A. baumannii BM2686. We conclude that aac(6')-I genes may be suitable for identification at the species level and for analysis of the phylogenetic relationships of Acinetobacter.