The effect of anisodamine on the structure of the gel phas and the properties of the acyl chain disordering transition of di~itoylphosphatidyi~yc-erol (DPPG) has been studied through high-sensitivity differential scanning calorimetry (DSC) and fluorescence polarization measurements of 16(9-anthroyloxyl)-pahnitic acid (16AP) and 3.(P-anthroyloxyl)-stearic acid (3AS), labeling, respectively, the ends and the third carbon of the acyl chains. The non-interdigitated DPPG multilamellar vesicles formed in HEPES buffer show clear fluidity gradient in their acyl chains, whereas the fluidity gradients are completely abolished in the presence of anisodamine. The DSC results showed that the phase transition temperature (T,,,) of DPPG is decreased and the enthalpy @I) is increased by anisodamine, while the pre-transition vanishes. At 3 mM anisodamine, the dH of DPPG reaches 9.6 kcal/mol. It can be concluded that DPPG forms an interdigitated gel phase in the presence of anisodamine. A molecular scheme for the interaction of anisodamine with DPPG is proposed.