Intramolecular excimer formation in pyrene-labeled phosphatidylcholine was used as a tool to determine thermodynamic characteristics of the lamellar to hexagonal phase transitions in a binary lipid system dilinoleoylphosphatidylethanolamine (DLPE)/palmitoyloleoylphosphatidylcholine (POPC). Upon an Lα/HII phase transition, the activation energy Ea for excimer formation increased from 5.6 ± 0.2 kcal/mol to 6.3 ± 0.2 kcal/mol, while the activation entropy ΔS‡ decreased from −40.0 ± 0.8 cal/K.mol to −38.4 ± 0.8 cal/K.mol. The results are consistent with the idea of molecular splaying of the acyl chains in the hexagonal phase. It is estimated that the molecular area at the terminal carbon of the lipid acyl chains increases by a factor of 2.2 upon the LαHII transition in DLPE/POPC.