The trabecular meshwork is continuously challenged by oxidants that are both present in the aqueous humor and generated within the tissue. In this study we have investigated the antioxidant properties of cultured calf trabecular meshwork cells and evaluated the ability of the compound 4-hydroxy-2,2,6,6-tetramethypiperidine 1-oxyl (TEMPOL), a superoxide dismutase mimic, to prevent H2O2-induced cell damage. The cells were found to possess a high level of reduced glutathione, an undetectable amount of oxidized glutathione, and significant activities of glutathione peroxidase, glutathione reductase, catalase, superoxide dismutase, glucose-6-phosphate dehydrogenase, and the hexose monophosphate shunt. The cells tolerated a 3-h exposure to a maintained, physiological level of H2O2 (0.02 mM); however, if the activity of glutathione reductase was inhibited, the same level of peroxide caused damage as indicated by cell contraction and blebbing. At a level of 0.05 mM H2O2, added to the medium as a single pulse, the shunt was stimulated eightfold and there were no significant effects on growth or morphology. However, a level of 0.1 mM H2O2 overwhelmed the antioxidant capability of the cells and produced severe effects. Treatment of the cells with TEMPOL prevented H2O2-induced inhibition of growth, formation of single-strand breaks in DNA, activation of the DNA-repair enzyme poly-ADP-ribose polymerase, and decrease in NAD, but TEMPOL was not able to prevent other changes such as the loss of GSH, decrease in glyceraldehyde-3-phosphate dehydrogenase activity, and stimulation of the shunt. Thus, certain intracellular effects of H2O2 in trabecular cells were shown to be caused directly by H2O2 whereas others were mediated through metal-catalyzed free radical reactions. The results indicate the presence of significant antioxidant activity in trabecular meshwork cells with a major contribution provided by the glutathione redox cycle.