In glaucoma, the increased release of glutamate is the major cause of retinal ganglion cell death. Cannabinoids have been demonstrated to protect neuron cultures from glutamate-induced death. In this study, we test the hypothesis that glutamate causes apoptosis of retinal neurons via the excessive formation of peroxynitrite, and that the neuroprotective effect of the psychotropic Delta9-tetrahydroxycannabinol (THC) or nonpsychotropic cannabidiol (CBD) is via the attenuation of this formation. Excitotoxicity of the retina was induced by intravitreal injection of N-methyl-D-aspartate (NMDA) in rats, which also received 4-hydroxy-2,2,6,6-tetramethylpiperidine-n-oxyl (TEMPOL,a superoxide dismutase-mimetic), N-omega-nitro-L-arginine methyl ester (L-NAME, a nitric oxide synthase inhibitor), THC, or CBD. Retinal neuron loss was determined by TDT-mediated dUTP nick-end labeling assay, inner retinal thickness, and quantification of the mRNAs of ganglion cell markers. NMDA induced a dose- and time-dependent accumulation of nitrite/nitrate, lipid peroxidation, and nitrotyrosine (foot print of peroxynitrite), and a dose-dependent apoptosis and loss of inner retinal neurons. Treatment with L-NAME or TEMPOL protected retinal neurons and confirmed the involvement of peroxynitrite in retinal neurotoxicity. The neuroprotection by THC and CBD was because of attenuation of peroxynitrite. The effect of THC was in part mediated by the cannabinoid receptor CB1. These results suggest the potential use of CBD as a novel topical therapy for the treatment of glaucoma.