Pseudomonas aeruginosa BM2656 was resistant to tobramycin and susceptible to gentamicin and amikacin by disk diffusion testing. This unusual resistance was not transferable by conjugation to Escherichia coli or P. aeruginosa PAO38, and plasmid DNA was not detected in this strain. A 0.9-kb fragment harboring the tobramycin resistance gene was cloned from BM2656 into pUC18, generating pAT129. Analysis for aminoglycoside-modifying activity in extracts of BM2656 and E. coli harboring pAT129 indicated that tobramycin resistance was due to synthesis of an aminoglycoside 6'-N-acetyltransferase type I [AAC(6')-I] enzyme which modified amikacin and tobramycin. Although amikacin was acetylated, the bactericidal synergism of this aminoglycoside with ceftazidime against BM2656 was minimally affected. The sequence of the DNA fragment was determined. It contained an aac (6')-Ib-like gene and was located downstream from a conserved region related to Tn21. The translated sequence of this aac(6')-Ib gene possessed 99.2% identity with the putative products of the aac(6')-Ib gene cassettes from Serratia marcescens and Klebsiella pneumoniae and 69% identity with the putative aacA(6')-II gene product from P. aeruginosa. We conclude that an aac(6')-Ib gene has spread to the chromosome of P. aeruginosa, probably by transposition.