This series of experiments systematically evaluated the effect of phospholipid headgroup structure on the interaction between rhodopsin and phospholipids. Two types of experiments were reported. First, ESR experiments involving spin-labeled phosphatidylserine, phosphatidic acid, and phosphatidylcholine demonstrated that, in the fluid-isotropic phase of dimyristoylphosphatidylcholine (DMPC)-rhodopsin membranes, the relative order of rhodopsin-induced immobilization was phosphatidic acid greater than phosphatidylcholine greater than phosphatidylserine. Second, the effect of rhodopsin incorporation on the dimyristoylphosphatidylserine (DMPS) gel to liquid-crystalline phase transition was analyzed with ESR techniques. A partial, binary phase diagram for the DMPS-rhodopsin system at pH 7.0 was constructed by studying the partitioning of Tempo between polar and hydrophobic domains as a function of temperature and system composition. A main result of this analysis was the finding that rhodopsin broadens and reduces the amplitude of the DMPS phase transition to a much smaller extent than it does the DMPC phase transition. When interpreted in terms of theoretical treatments of integral protein-lipid interactions, this indicates that rhodopsin has a lower affinity for DMPS than DMPC.