Ischemic preconditioning by a single event of ischemia and reperfusion (SIRPC) dramatically protects renal function against ischemia and reperfusion (I/R) induced several weeks later. We recently reported that reactive oxygen species (ROS) and oxidative stress were sustained in a kidney that had functionally recovered from I/R injury, thus suggesting an association between SIRPC and ROS and oxidative stress. However, the role of ROS in SIRPC remains to be clearly elucidated. In order to assess the involvement of reactive oxygen species (ROS) in SIRPC, mice were subjected to SIRPC (30 min of bilateral renal ischemia and 8 days of reperfusion), and then exposed to I/R injury. Thirty min of bilateral renal ischemia in the non-SIRPC mice resulted in a marked increase in plasma creatinine levels 4 and 24 h after reperfusion, which was not observed in the I/R in the SIRPC mice. SIRPC resulted in increases in the levels of kidney superoxide. Administrations of manganese(III) tetrakis(1-methyl-4-pyridyl) porphyrin (MnTMPyP, a cell-permeable superoxide dismutase (SOD) mimetic) and N-acetylcysteine (NAc, a ROS scavenger) to SIRPC-mice blocked the SIRPC-induced increase in superoxide levels, and removed approximately 48 to 64% of the functional protection of the SIRPC-kidney. Additionally, these administrations significantly inhibited I/R-induced increases of superoxide formation, hydrogen peroxide production, and lipid peroxidation, along with the inhibition of I/R-induced reductions in the expression and activity of manganese SOD (MnSOD), copper-zinc SOD (CuZnSOD), and catalase. Furthermore, administrations of MnTMPyP or NAc inhibited the SIRPC-induced increase of inducible nitric oxide synthase (iNOS) expression, but did not inhibit the SIRPC-induced increases in heat shock protein-25 (HSP-25) expression. In conclusion, the renoprotection afforded by SIRPC was triggered by ROS generated by SIRPC.