Potential of mean force (PMF), free energy perturbation (FEP), and molecular dynamics (MD) simulations were used to examine the association processes of K+ and Na+ with valinomycin in methanol. The estimated absolute free energy of binding of Na+ to valinomycin is similar to 1.1 to -2.5 kcal/mol, which is in reasonable agreement with the available experimental value of -1.2 +/- 0.4 kcal/mol. The calculated K+ free energies of association through the preferred HyV (isopropyl) face (the Lac (methyl, isopropyl) face was found to be disfavored) of valinomycin are -7.4 to -12.5 kcal/mol. This range of values overestimates the experimental free energy of binding (-6.4 +/- 0.4 kcal/mol) by similar to 1-6.1 kcal/mol, respectively. Graphical analysis and MD trajectories at various points of the PMF profiles show that the association/dissociation processes of K+ and Na+ are qualitatively similar, and the difference in binding affinity is based on the solvation free energy of the ions in methanol.