The thermotropic behavior of lipid vesicles prepared from dimyristoylphosphatidylcholine in the presence of cytochrome c oxidase has been studied by highly sensitive differential scanning microcalorimetry. This protein has a remarkable effect on the gel-liquid crystalline transition of dimyristoylphosphatidylcholine. In the presence of cytochrome c oxidase, the thermogram of the lipid vesicles exhibits a second endothermic peak, which is adjacent to the main lipid phase-transition peak and appears at a higher temperature. As the concentration of added protein increases, the two endothermic peaks become further separated, and the transition temperatures and the heats of transition corresponding to both endothermic peaks decrease. A greater decrease in the transition temperature at the lower-temperature peak with added protein suggests that the lower-temperature peak is more perturbed than the higher-temperature peak. The higher-temperature peak is not thermally reversible. Treatment of sample well above the transition temperature results in a reduction of the magnitude of the higher-temperature peak. The lipid-protein interaction contributing to the higher-temperature peak is discussed.