We studied the thermal response of 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphoethanolamine (POPE) by comparing the differential scanning calorimetry (DSC) data of liposomes with atomic force microscopy (AFM) observations on supported planar bilayers. Planar bilayers were obtained by using the Langmuir-Blodgett (LB) technique: the first leaflet transferred at 30 mN m -1 and the second at 25 mN m_1. The topographic evaluation of supported POPE bilayers above room temperature showed changes between 43.8 and 59.8 °C. These observations are discussed in relation to the main roughness (Ra) variations and are interpreted as the result of the lamellar liquid crystalline (Lα) to inverted hexagonal (HII) phase transition. High-magnification images obtained at 45 °C revealed intermediate structures in the transformation. Force spectroscopy (FS) was subsequently applied to gain further structural and nanomechanical insight into the POPE planar bilayers as a function of temperature. These measurements show that the threshold force (Fy), which is the maximum force, that the sample can withstand before breaking, increases from 1.91 ± 0.11 nN at 21 ° C up to 3.08 ± 0.17 nN at 43.8 °C. This behavior is interpreted as a consequence of the formation of intermediate structures or stalks in the transition from the Lα to HII phase. © 2008 American Chemical Society.