Glaucoma is a leading cause of irreversible world blindness. Oxidative damage and vascular injury have been implicated in the pathogenesis of this disease. The purpose of this study was to determine in human primary open angle glaucoma whether oxidative injury occurs in pre-laminar optic nerve blood vessels and glial cells. Following IRB approval, sections from post-mortem primary open angle glaucoma eyes (n=5) with mean age of 77 +/- 9 yrs (+/-SD) were compared to normal control eyes (n=4) with mean age 70 +/- 9 yrs (Eye Bank of Canada). Immunostaining with nitrotyrosine, a footprint for peroxynitrite-mediated injury, was performed and sections were double-labeled with markers for vascular endothelial cells, perivascular smooth muscle cells, and astrocytes with CD34, smooth muscle actin (SMA), and glial fibrillary acidic protein (GFAP), respectively. Immunostaining was captured in a masked fashion using confocal microscopy, and defined regions of interest for blood vessels and glial tissue. Intensity measurements of supra-threshold area in pixels as percent of the total number of pixels were calculated using ImageJ (NIH) and compared using two-tailed Mann-Whitney nonparametric tests between glaucoma and control groups. Colocalization coefficients with cell-specific markers were determined and compared with random coefficients of correlation. Increased nitrotyrosine immunoreactivity was observed in pre-laminar optic nerve head blood vessels of primary open angle glaucoma eyes compared to controls and this difference was statistically significant (1.35 +/- 1.11% [+/-SD] vs. 0.01 +/- 0.01%, P=0.016). NT-immunoreactivity was also increased in the glial tissue surrounding the pre-laminar optic nerve head in the glaucoma group and compared to controls, and this difference was statistically significant (18.37 +/-12.80% vs. 0.08 +/- 0.04%, P=0.016). Colocalization studies demonstrated nitrotyrosine staining in vascular endothelial and smooth muscle cells, in addition to astrocytes. Correlation coefficients for CD34, SMA, and GFAP were 0.37, 0.52, and 0.64, respectively. Oxidative injury is present in blood vessels and astrocytes in the pre-laminar optic nerve head in human primary open angle glaucoma. Peroxynitrite-mediated oxidative injury, whether primary or secondary, may contribute to the pathobiology of glaucoma disease.