We have investigated the thermotropic behavior of phosphatidylserine bilayers interacting with Mg 2+ either on one side or both sides, using differential scanning calorimetry. Large unilamellar vesicles (LUV) of phosphatidylserine exposed to Mg 2+ on the external side only displayed an upward shift of the gel-liquid transition temperature ( T m) of about 6–8 C° relative to the T m of LUV in Na +. Mg 2+ was shown not to enter the vesicle interior, by means of fluorescence measurements on encapsulated 8-hydroxyquinoline-5-sulfonate. Multilamellar vesicles prepared in the presence of Mg 2+, or vesicles prepared by Mg 2+-induced fusion of small unilamellar vesicles, had T m values that were shifted upward by about 16–17 C°. When the latter preparation was treated with EDTA to produce vesicles with Mg 2+ inside and Na + outside, the T m was found to be shifted again by only 6–8 C°. These observations indicate that the monolayer interacting with Na + fluidizes the monolayer interacting with Mg 2+, and thet the latter tends to solidify the former. The two monolayers thus appear to be coupled, possibly by hydrocarbon chain interdigitation.