Rat renal cortical mitochondria were isolated from Wistar male rats weighing 80 to 120 g to investigate whether the source of oxygen free radicals was renal cortical mitochondria enhanced by gentamicin. In renal cortical mitochondria with or without the addition of gentamicin, DMSO, DFO, CAT, SOD, and MTl were added separately, then incubated at 37°C for 90 min. Superoxide anions and hydroxyl radicals were then determined. The results showed that superoxide anions and hydroxyl radicals generated in mitochondria were enhanced by the addition of in vitro gentamicin (12.4 mg/mL) when compared to those without the addition of gentamicin. Dimethylsulfoxide (DMSO), catalase (CAT), and deferoxamine (DFO) significantly inhibited hydroxyl radicals enhanced by gentamicin, but superoxide dismutase (SOD) and metallothionein-1 (MTl) did not. SOD significantly inhibited the production of superoxide anions. Our data indicated that renal cortical mitochondria are the source of oxygen free radicals and that production is enhanced by gentamicin. This provides more insight on the pathogenetic role of hydroxyl radicals and superoxide anions in gentamicin-induced nephrotoxicity in vitro.