Complexation of valinomycin (VM) with the divalent cation Ca2+ in a lipophilic solvent, acetonitrile (CH3-CN), has been studied by using circular dichroism and proton and carbon-13 nuclear magnetic resonance (1H NMR and 13C NMR). From analyses of the spectral data, it is concluded that VM forms a 2:1 (peptide-ion-peptide) sandwich complex with Ca2+, at low concentration of VM. At moderate concentrations of the salt, in addition to the sandwich complex, an equimolar (1:1) complex different from those observed for potassium and sodium is also observed. At very large concentrations of the calcium salt, the data suggested a complex with a conformation similar to that of the free VM in polar solvents. Possible conformations for the sandwich and the equimolar VM-calcium complexes are proposed.