The thermodynamic phase behavior and trans-bilayer permeability properties of multilamellar phospholipid vesicles containing a short-chain DC₁₀PC phospholipid permeability enhancer have been studied by means of differential scanning calorimetry and fluorescence spectroscopy. The calorimetric scans of DC₁₄PC lipid bilayer vesicles incorporated with high concentrations of DC₁₀PC demonstrate a distinct influence on the lipid bilayer thermodynamics manifested as a pronounced freezing-point depression and a narrow phase coexistence region. Increasing amounts of DC₁₀PC lead to a progressive lowering of the melting enthalpy, implying a mixing behavior of the DC₁₀PC in the bilayer matrix similar to that of a substitutional impurity. The phase behavior of the DC₁₀PC-DC₁₄PC mixture is supported by fluorescence polarization measurements which, furthermore, in the low-temperature gel phase reveal a non-monotonic concentration-dependent influence on the structural bilayer properties; small concentrations of DC₁₀PC induce a disordering of the acyl chains, whereas higher concentrations lead to an ordering. Irreversible fluorescence quench measurements demonstrate a substantial increase in the trans-bilayer permeability over broad temperature and composition ranges. At temperatures corresponding to the peak positions of the heat capacity, a maximum in the trans-bilayer permeability is observed. The influence of DC₁₀PC on the lipid bilayer thermodynamics and the associated permeability properties is discussed in terms of microscopic effects on the lateral lipid organization and heterogeneity of the bilayer. Fluorescence polarization; Bilayer heterogeneity.