The interaction of ascorbate, hydrogen peroxide and oxygen has been examined in order to understand the equilibrium between these compounds that exists in the aqueous humor of the eye and their influence on function of the corneal endothelium. Ascorbate was found to promote corneal swelling when isolated corneas were perfused with a medium lacking glucose. This was found to be due to the rapid oxidation of ascorbate in the medium, yielding H2O2which is toxic to the endothelial cells. In the absence of oxygen, or if EDTA was added to the medium, no H2O2was produced from ascorbate, but ascorbate reacted with any pre-existing H2O2Oxidation of ascorbate in the aqueous humor is limited by the presence of glutathione (and, possibly, other compounds) and no significant increase in H2O2concentration occurs on standing in air. Nevertheless, the concentration of H2O2in the aqueous is directly dependent on the concentration of ascorbate secreted in the aqueous humor. Therefore, there must be a dynamic equilibrium in this fluid between ascorbate, H2O2and oxgyen, and it may be modulated by glutathione.Each of these substances is important in redox reactions, including free-radical production or scavenging. Consequently, when studying the effects on corneal or lenticular function of other agents which cause or relieve oxidant stress, it is critical that the modifying effects of ascorbate and H2O2, as they occur in vivo, be considered. A perfusion system is described which permits an approximation in vitro of stable concentrations of ascorbate, H2O2, SSH and O2similar to those found in the aqueous humor.