The bilayer stability of liposomes containing unsaturated phosphatidylethanolamine (PE) has been investigated by measuring the C~O and CH 2 stretching frequencies of the lipids at different lipid compositions and temperatures. Binary mixtures of 1-palmitoyl-2-oleoylphosphatidyleholine (POPC) and dilinoleoyl-PE (DLPE) are known to exhibit lamellar liquid crystalline (L,), inverted hexagonal (Hn) and metastable intermediate (I) phases. Abrupt increases in the C~--O and CH2 stretching frequencies at 65-75 and 90-95% PE, respectively, were found as the PE content of the DLPE/POPC mixtures was increased from 0 to 100%. These transitions were associated with the La-I and I-HII phase transitions of the DLPE/POPC mixtures, accordingly. The effects of three lipid perturbants, butylated hydroxytoluene (BHT), diacylglyeerol (DG) and cholesterol (CL), on the above L~-I and I-H n transitions were also examined. All perturbants were found to be effective in shifting the L~-I transition of the DLPC/POPC mixtures to a lower PE% as detected by the C=O stretching frequency measurements. On the other hand, the perturbants appeared to eliminate the I-Hn transition of the DLP~POPC mixtures as detected by the CH2 stretching frequency measurements. The effectiveness of the perturbants in promoting the La-I phase transition of the DLPE/POPC mixtures followed the order of DG > BHT > CL. The phase boundaries of this L~-I transition at different lipid and perturbant compositions were further quantitated by the use of three-dimensional contour plots (Cheng, Chem. and Phys. Lipids, 60, 119-125 (1991)) of the C~O frequency as a function of DLPE/POPC and perturbant/lipid compositions. It was concluded that the lipid C=O vibrational frequency is a useful and non-invasive parameter for examining the bilayer stability of liposomes.