The free energy governing K(+) conduction through gramicidin A channels is characterized by using over 0.1 micros of all-atom molecular dynamics simulations with explicit solvent and membrane. The results provide encouraging agreement with experiments and insights into the permeation mechanism. The free energy surface of K(+), as a function of both axial and radial coordinates, is calculated. Correcting for simulation artifacts due to periodicity and the lack of hydrocarbon polarizability, the calculated single-channel conductance for K(+) ions is 0.8 pS, closer to experiment than any previous calculation. In addition, the estimated single ion dissociation constants are within the range of experimental determinations. The relatively small free energy barrier to ion translocation arises from a balance of large opposing contributions from protein, single-file water, bulk electrolyte, and membrane. Mean force decomposition reveals a remarkable ability of the single-file water molecules to stabilize K(+) by -40 kcal/mol, roughly half the bulk solvation free energy. The importance of the single-file water confirms the conjecture of Mackay et al. [Mackay, D. H. J., Berens, P. H., Wilson, K. R. & Hagler, A. T. (1984) Biophys. J. 46, 229-248]. Ion association with the channel involves gradual dehydration from approximately six to seven water molecules in the first shell, to just two inside the narrow pore. Ion permeation is influenced by the orientation of the single-file water column, which can present a barrier to conduction and give rise to long-range coupling of ions on either side of the pore. Small changes in the potential function, including contributions from electronic polarization, are likely to be sufficient to obtain quantitative agreement with experiments.