The aim of this study was to investigate the relationship between antioxidant enzymes and reactive oxygen species production in diapausing larvae of the European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae) kept at 5°C, -3°C and -16°C for two weeks. The amount of hydrogen peroxide (H2O2), activity of antioxidant enzymes, copper zinc superoxide dismutase (CuZnSOD), manganese superoxide dismutases (MnSOD) and catalase (CAT) in whole body homogenates, as well as the electron paramagnetic resonance (EPR) spectroscopy of this insect's whole body were analysed. A higher level of melanin radical and lower CuZnSOD and CAT activities were found in larvae kept at -3°C than at 5°C and -16°C. At the same temperature (-3°C) an elevated H2O2 concentration was recorded. A possible regulatory role of H2O2 at -3°C, which is the temperature that triggers freezing tolerance, is suggested.