We have investigated the molecular features of recombinant membranes that are necessary for the photochemical function of rhodopsin. The magnitude of the metarhodopsin I to metarhodopsin II phototransient following a 25% ± 3% bleaching flash was used as a criterion of photochemical activity at 28 °C and pH 7.0. Nativelike activity of rhodopsin can be reconstituted with an extract of total lipids from rod outer segment membranes, demonstrating that the protein is minimally perturbed by the reconstitution protocol. Rhodopsin photochemical activity is enhanced by phosphatidylethanolamine head groups and docosahexaenoyl (22:6ω3) acyl chains. An equimolar mixture of phosphatidylethanolamine and phosphatidylcholine containing 50 mol % docosahexaenoyl chains results in optimal photochemical function. These results suggest the importance of both the head-group and acyl chain composition of the rod outer segment lipids in the visual process. The extracted rod lipids and those lipid mixtures favoring the conformational change from metarhodopsin I to II can undergo lamellar (LJ to inverted hexagonal (H11) phase transitions near physiological temperature. Interaction of rhodopsin with membrane lipids close to a Lα to H11(or cubic) phase boundary may thus lead to properties which influence the energetics of conformational states of the protein linked to visual function.