Nanosized ordered domains formed in 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DOPC/DPPC) and DOPC/cholesterol (Ch) liposomes were characterized using a newly developed (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) quenching method. The membrane fluidity of the DOPC/DPPC liposomes, evaluated by the use of 1,6-diphenyl-1,3,5-hexatriene (DPH), increased significantly above their phase-transition temperature. The fluorescence spectra of 6-lauroyl-2-dimethylamino naphthalene (Laurdan) indicated the formation of an immiscible ordered phase in the DOPC/DPPC (50/50) liposomal membrane at 30 degrees C. The analysis of the membrane polarity indicated that the surface of the liquid-disordered phase was hydrated whereas that of the ordered phase was dehydrated. DOPC/DPPC and DOPC/Ch (70/30) Liposomes exhibited heterogeneous membranes, indicating that nanosized ordered domains formed on the surface of the DOPC/DPPC liposomes. The size of these nanosized ordered domains was estimated using the TEMPO quenching method. Because TEMPO can quench DPH distributed in the disordered phases, the remaining fluorescence from DPH is proportional to the size of the ordered domain. The domain sizes calculated for DOPC/DPPC (50/50), DOPC/DPPC (25/75), DOPC/Ch (70/30), and DOPC/DPPC/Ch (40/40/20) were 13.9, 36.2, 13.2, and 35.5 angstrom, respectively.