To examine the expression of phosphorylated c-Jun N-terminal kinase (JNK) in cells in the retinal ganglion cell layer of glaucoma, intraocular pressure (IOP) of adult Wistar rats was elevated unilaterally by repeated trabecular argon laser photocoagulation 5 days after intracameral injection of India ink. Animals were euthanized after 3 days, and 1, 2, and 5 weeks of IOP elevation. Immunohistochemistry with specific antibodies against phosphorylated JNK was performed on retinas. Retrograde labeling using Fluorogold and fluorescence immunohistochemistry was performed on retinas 5 weeks after IOP elevation. TdT-mediated biotin-dUTP nick end labeling (TUNEL) was performed on the retinal sections to determine the rate of cell death. There was increased IOP (52.3%) from 3 days to 5 weeks after repeated trabecular laser photocoagulation. Mean number of TUNEL-positive cells in the retinal ganglion cell layer of eyes with experimental glaucoma was 0.43, 0.36, 0.57, and 0.19 per retinal section at 3 days, and 1, 2 and 5 weeks, respectively. No TUNEL-positive cells were noted in controls. In parallel to TUNEL, significantly increased numbers of phosphorylated JNK-labeled cells in the retinal ganglion cell layer were noted at 3 days (9.95 versus 4.15; P=0.005), 1 week (7.65 versus 4.00; P=0.006), 2 weeks (9.13 versus 4.48; P=0.032), and 5 weeks (8.06 versus 4.96; P=0.017) of IOP elevation when compared with contralateral control eyes. Fluorogold labeled RGCs were co-localized with increased phosphorylated JNK immunoreactivity. Some TUNEL-positive cells were phosphorylated JNK immuno-positive. Phosphorylation of JNK occurs in experimental glaucoma and may play a role in retinal ganglion cell death.