The use of gel filtration chromatography with Sephadex as a separation medium was used in order to study flunitrazepam (FNTZ) partitioning into artificial model membranes consisting of dipalmitoyl‐phosphatidylcholine (dpPC) vesicles, under controlled temperature conditions. In this system two phenomena are taking place simultaneously: the ligand–liposome interaction and the lipid self‐aggregation to form the liposome. The liposome–FNTZ interaction was evidenced by the non‐enantiography of the first derivative of FNTZ elution peak in frontal chromatography through Sephadex G‐75. On the other hand, the presence of FNTZ reduced liposomes mean size and increased their size dispersion as evidenced by molecular filtration through Sephadex G‐200. The dpPC–buffer FNTZ partition coeficient determined in zonal chromatography through Sephadex G‐10 increased about 33% when the temperature rose above the temperature of dpPC transition from the liquid crystalline to the fluid phase. Gel filtration chromatography seems a suitable technique to study lipid liposome–FNTZ interactions at a qualitative level. In addition, this technique has the advantage over other methods of giving the possibility of observing the mutual effects exerted between the drug and the self‐aggregating structure. © 1997 John Wiley & Sons, Ltd.