In the present study, we investigated the generation of lipid peroxides and changes in total phospholipid levels in the kidney tissue of rats with acquired resistance to gentamicin (GM). GM resistance was induced in Sprague-Dawley male rats by subcutaneously administering each rat a dose of 80 mg/kg/day of GM for 40 consecutive days. Twelve days after the GM administration, serum urea nitrogen peaked at 35 mg/dl. The urinary creatinine excretion progressively decreased, beginning 4 days after the start of GM administration. The fractional excretion of sodium progressively increased, beginning 4 days after the start of GM administration, peaking on the 10th day. However, despite the continuation of GM administration, the urinary creatinine excretion gradually increased, and the serum urea nitrogen concentrations recovered to previous levels after 21 days. We also analyzed the relationship between the acquired resistance to GM and changes in the reduced glutathione content and glutathione peroxidase activity. Simultaneously, we investigated sequential changes in the activities of phospholipase A<sub>2</sub> and phospholipase C, which release peroxidized membrane phospholipids into the cytoplasm via hydrolysis, as well as the relationship between changes in the kidney tissue phospholipid composition (sphingomyelin/phosphatidylcholine ratio) and renal function. We found that (1) the kidney tissue glutathione content rapidly decreased after GM administration before subsequently increasing and being maintained at a higher level; (2) the glutathione peroxidase activity showed a persistent decrease after GM administration; (3) the kidney tissue phospholipase A<sub>2</sub> activity decreased after GM administration, while the phospholipase C activity exhibited a sustained increase from 21 days, and (4) the spingomyelin/phosphatidylcholine ratio decreased on the 4th day before stabilizing when acquired resistance was obtained. Based on these findings, we conclude that an increased supply of reduced glutathione and the induction of an antioxidase, substituting for glutathione peroxidase, may play a significant role in the acquisition of resistance to acute renal failure which occurs with continuous GM administration. Improved membrane fluidity, achieved by maintenance of the membrane phospholipid composition by increased phospholipase C activity, may be an additional factor contributing to the recovery of the renal function.