The purpose of the study was to determine the in vitro resistance of Gram-negative bacilli to isepamicin, a new aminoglycoside. Resistance rates of Gram-negative bacilli to isepamicin was determined with NCCLS disk diffusion method and compared with those of other four aminoglycosides. Totally 332 strains including 63 Acinetobacter spp., 70 Pseudomonas aeruginosa, 97 Escherichia coli, 61 Klebsiella pneumoniae 16 Proteus mirabilis and 25 Serratia marcescens strains were studied. Resistance rate was 43.3% to gentamicin (G), 39.4% to tobramycin (T), 28.6% to netilmicin (N), 24.7% to amikacin (A) and 20.7% to isepamicin (I). Resistance to G, T, N, A, and I was 73.0%, 49.2%, 41.2%, 68.2%, 71.4% for Acinetobacter spp.; 48.5%, 44.2%, 47.0%, 35.7%, 42.8% for P. aeruginosa, 20.6%, 30.0%, 6.1%, 1.0%, 0% for E. coli; 60.6%, 57.3%, 44.2%, 13.1%, 6.5% for K. pneumoniae 12.5%; 12.5%, 12.5%, 6.2%, 0% for P. mirabilis 44.0%, 44.0%, 20.0%, 24.0%, 4.0% for S. marcescens, respectively. These results support the idea that isepamicin could be used as an alternative aminoglycoside especially in the regions where resistance to the other aminoglycosides has increased.