Proton transfer reactions of (M + 2H)(2+) and (M + H + X)(2+) ions (X = Li, Na, K) of gramicidin S with four reference bases are measured. From the kinetics of proton transfer, the apparent gas-phase basicities of the (M + H)(+) and (M + X)(+) ions are assigned values of 219.1 +/- 2.8 and 223.3 +/- 2.4 kcal/mol, respectively. We attribute the higher apparent basicity of the (M + X)(+) ions to a larger separation between charges. From this difference in basicities is calculated a charge separation distance of similar to 11.5 Angstrom in the (M + H + X)(2+) ions using a simple point charge model. This value is consistent with a structure in which the alkali metal ion is attached to the exterior surface of the peptide and is in excellent agreement with structures obtained by molecular modeling. Rates of proton transfer from (M + H + X)(2+) to dipropylamine decrease as the size of the alkali metal ion increases. This indicates that the binding site is the same for each of these metal ions.