Aminoglycoside 2''-O-nucleotidyltransferase (AAD(2'')) mediates bacterial resistance to dibekacin, gentamicin, kanamycin, sisomicin and tobramycin. Its coding sequence, aadB, is part of Tn21-related transposon, Tn4000. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 177 amino acids with a calculated molecular weight of 21,240. The predicted amino acid sequence revealed up to 27% homology to that of three nucleotidyltransferases of type AAD(3''), which are widely distributed among Gram-negatives, and to the AAD(9) from Staphylococcus aureus transposon Tn554. The regions flanking aadB suggest that its insertion into Tn21 arose from a site-specific recombination event adjacent to the aadA gene.