Pharmacological experiments have suggested that ocular ischemia, induced by high intraocular pressure in the rabbit, provokes an oxidative stress responsible for functional alteration of the retina. However, the nature of the oxidant chemical species and their mode of generation were not elucidated. The aim of the present studies was to characterize the oxygen-derived free radicals produced during and/or after the hyperpressure period. The technique used was based on electron spin resonance spin trapping analysis of the signals obtained in microdialysates of the retina perfused with the nitrone 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). The oxidative stress was also evaluated under ischemia and reperfusion periods by measuring the level of ascorbate in the retina via electron spin resonance detection of the ascorbyl free radical-dimethyl sulfoxide (AFR-DMSO) complex. Electroretinograms were recorded to determine the functional consequences of high intraocular pressure and free radical generation. Our results show that superoxide dismutase-inhibitable DEPMPO/hydroxyl radical adducts were generated during the high intraocular pressure period and that the oxidative stress was not increased at reperfusion as assessed by spin trapping and AFR-DMSO measurements. Functional protection provided by free radical scavengers (superoxide dismutase+catalase, TEMPO nitroxide+catalase and dimethylthiourea) against high intraocular pressure-induced electroretinogram alteration confirmed these observations. In conclusion, these experiments demonstrate for the first time by direct measurement that oxygen-derived free radicals are produced by the retina during acute ischemia. This generation could be the explanation for electroretinogram alteration.