Phase behavior of hydrated bilayer of binary phospholipid mixtures composed of 1,2-distearoylphosphatidylcholine (DSPC) and 1-stearoyl-2-oleoylphosphatidylcholine (SOPC), and DSPC and 1-oleoyl-2-stearoylphosphatidylcholine (OSPC) was examined in terms of a pseudo-binary phase diagram. The phase boundaries determined by differential scanning calorimetry (DSC) were analyzed based on a thermodynamic model applying the Bragg–Williams approximation for non-ideality of mixing. The interchange energy parameters derived from this approach for liquid-crystalline and solid-like gel phases were ρ 0 (L)=2.0 kJ mol −1 and ρ 0 (S)=5.4 kJ mol −1 for SOPC/DSPC mixture and ρ 0 (L)=1.9 kJ mol −1 and ρ 0 (S)=5.5 kJ mol −1 for OSPC/DSPC mixture. These values for both mixture systems are close to each other, and also close to those obtained for dimyristoylphosphatidylcholine (DMPC)/DSPC mixture. This suggests that the effect of introducing a cis-unsaturated acyl chain into a diacylphosphatidylcholine on the mixing behavior with homologous phospholipid with two identical saturated acyl chains is comparable to that of shortening the length of saturated acyl chain by four methylene units.