Reduced and overoxidized forms of peroxiredoxins (Prxs) in vivo were assessed in cultured human umbilical vein endothelial cells (HUVEC) and in damaging light-exposed mouse retinal tissues by two-dimensional (2D) gel electrophoresis and Western blot analysis. Acidic isoelectric point (pI) shift of protein spots of Prx2, 3, 4, and 6, which indicate formation of overoxidized Prx was clearly observed after treatment with H(2)O(2) but was not observed after treatment with the radical initiators 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) and 2,2'-azobis [2-(2-imidazolin-2-yl)propane] dihydrochloride (AIPH); this finding indicated selective oxidation of Prx molecules by H(2)O(2). Following the exposure of the eyes of mice to visible light (5000 lx) for up to 4h, no acidic spot shift was observed in Prx1, 2, and 6 in both retinal and retinal pigment epithelial samples; this finding indicated the possible involvement of the photosensitization reaction rather than free radicals derived from H(2)O(2) in this pathology. Detection of overoxidized Prxs may be applicable for assessing the mechanisms of cell/tissue damage caused by oxidative stress in vivo.