The interaction of N-myristoylethanolamine (NMEA) with cholesterol is investigated by differential scanning calorimetry (DSC), fast-atom-bombardment mass spectrometry (FAB-MS) and computational modelling. Addition of cholesterol to NMEA leads to a new phase transition at 55°C besides the chain-melting transition of NMEA at 72.5°C. The enthalpy of the new transition increases with cholesterol content up to 50 mol%, but decreases thereafter, vanishing at 80 mol%. The enthalpy of the chain-melting transition of NMEA decreases with an increase in cholesterol; the transition disappears at 50 mol%. FAB-MS spectra of mixtures of NMEA and cholesterol provide clear signatures of the formation of {[NMEA+cholesterol]+} {[NMEA+cholesterol+Na]+}. These results are consistent with the formation of a 1:1 complex between NMEA and cholesterol. Molecular modelling studies support this experimental finding and provide a plausible structural model for the complex, which highlights multiple H-bond interactions between the hydroxy group of cholesterol and the hydroxy and carbonyl groups of NMEA besides appreciable dispersion interaction between the hydrocarbon domains of the two molecules. © 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.