The cytotoxic effect of peroxynitrite on cerebellar granule neurons was studied. Exposure of cerebellar granule neurons to 10 &mgr;mol/L peroxynitrite triggered apoptosis in vitro, which was confirmed by both morphological (nuclear morphology observed by fluorescence microscopy) and biochemical evidence (DNA fragmentation detected by ELISA). Using ESR spin labeling technique, the alteration of biophysical characteristics of neuronal cell membrane during the apoptotic process was studied. The results indicate that after treatment with peroxynitrite, the fluidity of both the surface layer and the deep layer of the neuronal cell membrane decreased markedly, and the S/W ratio of the membrane protein thiol groups increased significantly. Pre-treating cerebellar granule neurons with antioxidant EPC-K1, a novel water-soluble derivative of vitamin C and vitamin E, alleviated the oxidative injury and prevented cells from apoptosis.