The six possible phosphatidylcholines containing two different chains derived from myristic, palmitic, and stearic acids were synthesized, and their bilayer structures were investigated by high-sensitivity differential scanning microcalorimetry. Chain migration during the syntheses caused each of the lipids to contain about 10% of the corresponding positional isomer. A phase diagram for each pair of isomers was constructed to permit estimation of the transition properties of the pure mixed-length phospholipids. The phase transitions of these lipids were found to be similar to those of saturated like-chain phosphatidylcholines. The main transition temperatures and enthalpies fall within the range of those for the like-chain lipids. In each pair of positional isomers, the isomer having the longer chain at position 2 on the glycerol backbone has the higher transition temperature and enthalpy. The transition curves of the pure mixed-chain lipids with myristic acid at position 2 and either palmitic or stearic acid at position 1 exhibited two partially separated peaks for the main transition. No satisfactory interpretation of this unexpected phenomenon has been developed.