Amikacin has been very useful in the treatment of infections caused by multiresistant bacteria because it is refractory to the actions of most modifying enzymes. However, the spread of AAC(6')-I-type acetyltransferases, enzymes capable of catalyzing inactivation of amikacin, has rendered this antibiotic all but useless in some parts of the world. The aminoglycoside 6'-N-acetyltransferase type Ib, which is coded for by the aac(6')-Ib gene, mediates resistance to amikacin and other aminoglycosides. RNase H mapping and computer prediction of the secondary structure led to the identification of five regions accessible for interaction with antisense oligodeoxynucleotides in the aac(6')-Ib mRNA. Oligodeoxynucleotides targeting these regions could bind to native mRNA with different efficiencies and mediated RNase H digestion. Selected oligodeoxynucleotides inhibited AAC(6')-Ib synthesis in cell-free coupled transcription-translation assays. After their introduction into an Escherichia coli strain harboring aac(6')-Ib by electroporation, some of these oligodeoxynucleotides decreased the level of resistance to amikacin. Our results indicate that use of antisense compounds could be a viable strategy to preserve the efficacies of existing antibiotics to which bacteria are becoming increasingly resistant.