The duration of hydrogen peroxide (H2O2) exposure was examined in confluent monolayers of human corneal endothelium (HCE) and human lens epithelium (HLE) by comparing 3-minute, 15-minute, 30-minute, and 90-minute treatment protocols. The cells showed remarkable ability to withstand high doses of H2O2 (1 mM, approximately 30 ppm) for up to 30 minutes without noticeable morphologic alterations or decreases in rubidium-86 chloride (86RbCl) uptake (a marker used to evaluate damage to the cells' active transport properties). In contrast, when H2O2 levels were maintained for 90 minutes, a substantially lower dose (0.1 mM, approximately 3 ppm) produced cell death, morphologic alterations, and depression of 86RbCl uptake. The marked difference in the dose-response for injury also appeared to be associated with a decrease in cells' abilities to remove H2O2 from the tissue culture medium. Knowledge of the time-dependency for H2O2 damage may be useful in the design of future in vitro studies concerning H2O2 effects, and may help explain the apparent lack of major cellular damage in vivo if topical H2O2 solutions are removed rapidly from the eye.