Fourier transform infrared spectroscopy was used to characterize the stretching vibrations of the ester carbonyl groups and the amide I vibration of the amide group of N-acylphosphatidylethanolamine (N-acylPE) bilayers. To obtain quantitative information about changes in hydration, band profiles were simulated with Gaussian-Lorentzian functions. The results are particularly significant since they probably reflect different changes in the hydrogen-bonding interactions in the polar/apolar interfacial region at the gel-liquid-crystalline phase transition of the N-acylPE bilayers related to the N-acyl chain length. In the same way, the carbonyl ester interfacial region of N-acylPEs is spectroscopically similar to those of phosphatidylethanolamine bilayers, in which two populations of hydrogen-bonded ester carbonyl groups were observed. Moreover, our study provides additional data pertinent to the physical basis of the thermotropic phase behaviour of aqueous dispersions of N-acylPEs and the basis of the differences between these bilayers and the more intensively studied phosphatidylcholine and phosphatidylethanolamine bilayers.