We have developed an in vitro system in which lipid peroxidation can be produced in a predictable fashion and have studied the effects of peroxidation on regenerability of rhodopsin in rod outer segments (ROS). ROS isolated by sucrose flotation from dark-adapted retinas of Rana pipiens were suspended in various concentrations of FeSO4 and 1 or 2 mM ascorbic acid for 10 minutes. An increase in lipid hydroperoxides (measured as conjugated dienes) and a decrease in 22:6 omega 3 were determined for each Fe+2 concentration and used as an index of peroxidation. Following incubation with and without FeSO4, ROS were pelleted and rhodopsin was regenerated with 15 micron 11-cis-retinal. The regenerability of rhodopsin in ROS in which significant lipid peroxidation had taken place was reduced by 40-50% compared to controls. DTPA (diethylenetriamine pentaacetic acid), EDTA, and EGTA protected against lipid peroxidation by Fe+2 and allowed almost complete regeneration of rhodopsin. Incubation with DTPA also prevented the destruction of vitamin E in ROS. Other compounds containing primary amine groups did not protect against peroxidation or loss of regenerability. These experiments indicate that lipid peroxidation is associated with a loss of regenerability of the visual pigment rhodopsin.