AbstractVarious nanoscopic channels of roughly equal diameter and length facilitate single-file diffusion at vastly different rates. The underlying variance of the energetic barriers to transport is poorly understood. First, water partitioning into channels so narrow that individual molecules cannot overtake each other incurs an energetic penalty. Corresponding estimates vary widely depending on how the sacrifice of two out of four hydrogen bonds is accounted for. Second, entropy differences between luminal and bulk water may arise: additional degrees of freedom caused by dangling OH-bonds increase entropy. At the same time, long-range dipolar water interactions decrease entropy. Here, we dissect different contributions to Gibbs free energy of activation, ΔG‡, for single-file water transport through narrow channels by analyzing experimental results from water permeability measurements on both bare lipid bilayers and biological water channels that (i) consider unstirred layer effects and (ii) adequately count the channels in reconstitution experiments. First, the functional relationship between water permeabilities and Arrhenius activation energies indicates negligible differences between the entropies of intraluminal water and bulk water. Second, we calculate ΔG‡ from unitary water channel permeabilities using transition state theory. Plotting ΔG‡ as a function of the number of H-bond donating or accepting pore-lining residues results in a 0.1 kcal/mol contribution per residue. The resulting upper limit for partial water dehydration amounts to 2 kcal/mol. In the framework of biomimicry, our analysis provides valuable insights for the design of synthetic water channels. It thus may aid in the urgent endeavor towards combating global water scarcity.