We have examined the role of reactive oxygen metabolites (ROM) in gentamicin nephrotoxicity and in glycerol-induced acute renal failure, a model for myoglobinuric acute renal failure. Several agents which affect mitochondrial respiration have been shown to enhance the generation of hydrogen peroxide. Based on gentamicin s ability to alter mitochondrial respiration both in vitro and in vivo we postulated that gentamicin may enhance the generation of ROM by renal com'cal mitochondria. Gentamicin, in a dose-dependent farhion, enhanced hydrogen peroxide production by rat renal cortical mitochondria as measured by the decrease in scopoletin jluorescence. At the highest concentration of gentamicin tested (4.0 mM), the rate of hydrogen peroxide generation w s markedly increased from 0. I7 f 0.02 to 6.21 f 0.67 nmol/mg/m*n. We next demonstrated that hydroxyl radical scavengers and an iron chelator provide a marked functional and histological protection in gentamicin-induced acute renal failure in rats. Hydroxyl radical scavengers and the iron chelator deferoxamine also protected renal function in glycerol-injected rats, a model for acute renal failure due to muscle injury. Although these data suggest that ROM may be important mediators of toxic renal injury, in vivo generation of ROM by kidney in normal and pathological states has not been previously examined. Aminotriazole (An irreversibly inactivates catalase only in the presence of hydrogen peroxide and previous studies have shown that AT-medated inhibition of catalase in a sensitive measure of in vivo changes in the hydrogen peroxide generation. Using this method, we have demonstrated the in vivo generation of hydrogen peroxide under normal conditions and enhanced generation of hydrogen peroxide in rats treated with gentamicin or glycerol. Finally, in in vitro studies we have shown that iron and intracellular calcium play a critical role in hydrogen peroxide-mediated cytotoxicity to LLC-PK, cells, a renal tubular epithelial cell line. Taken together our data provide evidence for a role of ROM in gentamicin and glycerol-induced acute renal failure and provide evidence for the role of iron and calcium in oxidant injury to renal tubular epithelial cells.