Biophysical measurements of multidrug transporters in vitro can often be of limited relevance to the natural in vivo behavior. In particular, the properties of transporters when removed from their native bilayer and solubilized in detergents or lipids can differ significantly from their in vivo properties, reducing the value of in vitro measurements for the design of antagonists to the transporters. This problem can be addressed by studying the transport protein in liposomes in which the properties of the liposome bilayer are altered through systematic changes in lipid composition. Isothermal titration calorimetry can be used to determine the properties of the lipid-reconstituted protein in bilayers of different lipid compositions as well as to quantify the percentage recovery of functional protein in different lipids. Both these measurements lead to an accurate analysis of substrate binding activity. The approach is illustrated here for the small multidrug transport protein, EmrE from Escherichia coli . The percentage of functional EmrE successfully reconstituted into liposome depends on lipid composition. Differences in ligand binding and subtle differences in the secondary structure also occur in different lipid compositions.