Earlier studies have suggested the formation of a 1:1 (mol/mol) complex between N-myristoylethanolamine (NMEA) and cholesterol in aqueous dispersion. In this study, this interaction has been investigated further by differential scanning calorimetry (DSC) on dry mixtures of NMEA, N-palmitoylethanolamine (NPEA) and N-stearoylethanolamine (NSEA) with cholesterol. The results obtained indicate that addition of cholesterol to NMEA leads to a new phase transition at 86.5°C, besides the solid-liquid phase transition of NMEA at 95°C. The intensity of the peak corresponding to the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, whereas the intensity of the peak corresponding to the melting of NMEA decreases with increasing cholesterol content, with concomitant and gradual shift to lower temperatures and vanishes at 50 mol% cholesterol. These results are consistent with the formation of a 1:1 molar complex between NMEA and cholesterol proposed earlier and indicate that these two amphiphiles are associated in the solid state as well. DSC studies on hydrated mixtures of NPEA and NSEA with cholesterol yielded results that parallel those obtained with the NMEA/cholesterol system, indicating that these two long-chain NAEs also form 1:1 (mol/mol) complexes with cholesterol.