The thermal phase behaviors of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) large unilamellar vesicles (LUVs) and multilamellar vesicles (MLVs) were compared by fluorescence spectroscopy, using PPDPC (1-palmitoyl-2[10-(pyren-1- yl)]decanoyl-sn-glycero-3-phosphocholine) as a reporter, in parallel with differential scanning calorimetry (DSC). A striking difference is seen between MLVs and LUVs in the lateral organizational dynamics of PPDPC, in particular, below the main phase transition temperature T_m, with efficient clustering of PPDPC into fluid microdomains in the L_β′ and P_β′ (ripple) phases of DPPC MLVs. In the P _β′ phase of MLVs, the probe is likely to become enriched in linear line defects, restricting intermolecular collisions to occur in a quasi one-dimensional system. In contrast, fluorescence and DSC data both suggest that the P_β′ phase is not well-defined in LUVs. Fluorescence anisotropy for 1-palmitoyl-2-[3-(diphenylhexatrienyl)propanoyl]-sn- glycero-3-phosphocholine (DPH-PC) revealed similar acyl chain order for both LUVs and MLVs in the L_β′ and P_β′ phases. However, for MLVs with this probe, T_m determined from anisotropy was elevated by 0.7°, with higher anisotropy evident in the L_α phase compared to LUVs. These differences in the thermal phase behavior of the two types of liposomes are likely to derive from the augmented acyl chain order due to cooperative coupling of the lamellae of DPPC MLVs, thus manifesting in new, emerging material properties in the latter type of bilayer membrane assembly, as reflected in the organizational dynamics of the pyrene-labeled analogue.