The subacute toxicity in the dog of isepamicin (HAPA-B), a new aminoglycoside antibiotic, was studied in comparison with amikacin (AMK). HAPA-B at dose levels of 6.25, 25 and 100 mg/kg/day and AMK at dose levels of 25 and 100 mg/kg/day were given intramuscularly for 30 days. Only one female dog in the AMK 100 mg/kg dose group died on day 29. The activities of urinary NAG and gamma-GTP increased dose-relatedly in dogs in the 25 and 100 mg/kg dose groups of either drug. Discoloration and/or enlargement of the kidneys were observed in dogs at 100 mg/kg of either drug. Hemorrhage and/or edema at the injection sites were observed in dogs in the 25 and 100 mg/kg dose groups of either drug. Increases of absolute and relative kidney weights were observed in the AMK 100 mg/kg group. An increase of relative kidney weights was also observed in the HAPA-B 100 mg/kg group. In microscopic observations, various histopathological changes of renal tubules and tubular epithelium were observed dose-relatedly in the 25 and 100 mg/kg groups of both drugs. At a dose of 25 mg/kg, there was no significant difference of the incidence and severity of these renal changes between HAPA-B and AMK groups. Desquamation and necrosis of the renal tubular epithelium were observed only in the AMK 100 mg/kg group. The necrosis in these changes was considered to be one of the severest possible changes in renal tubules, hence the renal toxicity of HAPA-B was judged to be milder than that of AMK at the dose level of 100 mg/kg. The non-toxic dose of HAPA-B in this study was estimated to be 6.25 mg/kg.