The bilayer stability of liposomes containing unsaturated phosphatidylethanolamine (PE) has been investigated by measuring the CO and CH2 stretching frequencies of the lipids at different lipid compositions and temperatures. Binary mixtures of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) and dilinoleoyl-PE (DLPE) are known to exhibit lamellar liquid crystalline (Lα), inverted hexagonal (HII) and metastable intermediate (I) phases. Abrupt increases in the CO 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 Lα-I and I-HII phase transitions of the DLPE/POPC mixtures, accordingly. The effects of three lipid perturbants, butylated hydroxytoluene (BHT), diacylglycerol (DG) and cholesterol (CL), on the above Lα-I and I-HII transitions were also examined. All perturbants were found to be effective in shifting the Lα-I transition of the DLPC/POPC mixtures to a lower PE% as detected by the CO stretching frequency measurements. On the other hand, the perturbants appeared to eliminate the I-HII transition of the DLPE/POPC mixtures as detected by the CH2 stretching frequency measurements. The effectiveness of the perturbants in promoting the Lα-I phase transition of the DLPE/POPC mixtures followed the order of DG > BHT > CL. The phase boundaries of this Lα-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 CO frequency as a function of DLPE/POPC and perturbant/lipid compositions. It was concluded that the lipid CO vibrational frequency is a useful and non-invasive parameter for examining the bilayer stability of liposomes.