The binding of a peptide to a biological membrane is often accompanied by a transition from a random coil structure to an amphipathic alpha-helix. Recently, we have presented a new approach which allows the determination of the thermodynamic parameters of membrane-induced helix formation [Wieprecht et al. (1999) J. Mol Biol. 294, 785]. It involves a systematic variation of the helix content of a given peptide by double D-substitution and a correlation of the binding parameters with the helicity. Here we have used this method to study membrane-induced helix formation for the presequence of rat mitochondrial rhodanese (RHD). The thermodynamic parameters of binding of the peptide RHD and of four of its double D-isomers were determined for 30 nm (SUVs) and 100 nm (LUVs) unilamellar vesicles composed of phosphatidylcholine/phosphatidylglycerol (3:1) using circular dichroism spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry. The incremental changes of the thermodynamic parameters of helix formation were found to be very similar for SUVs and LUVs. Membrane-induced helix formation of RHD entailed a negative enthalpy of Delta H(helix) = -0.5 to -0.6 kcal/mol/residue and was opposed by an entropy of about Delta S(helix) = -1 to -1.4 cal/mol K/residue. The free energy of helix formation, Delta G(helix), was about -0.2 kcal/mol, and helix formation accounted for 50-60% of the total free energy of membrane binding. Dye-release experiments were used to assess the role of helix formation for the membrane perturbation potential of the peptides. While helix formation plays a major role for membrane binding, it appears to have little importance for inducing membrane leakiness.