The aacA1 gene, which encodes a 6'-N-acetyltransferase [AAC(6')-I] mediating resistance to kanamycin, tobramycin, and amikacin, was cloned from the Citrobacter diversus R plasmid pBWH100 into the Escherichia coli vector pBR322. The complete nucleotide sequence of the gene and flanking regions was determined. A protein of approximately 21 kilodaltons was identified when the chimeric plasmid encoding the aacA1 gene was introduced into E. coli maxicells. This value is consistent with the size predicted for a protein translated from the open reading frame of the gene.