The structure and interactions of water species in hydrated Nafion membranes as a function of water content were investigated on the basis of medium-infrared spectral analysis and molecular dynamics (MD) simulations. The spectral decomposition of the FT-IR data in the stretching OH region was performed on different levels of hydration of the sulfate functional groups (λh2o/rso3- = 2-22). Quantum mechanical calculations of two model systems [perfluoroethanesulfonic acid/(H 2O)6 cluster] and a [perfluorobutanesulfonic acid/(H 2O)6 crystal] were carried out in order to account for the band assignments of Nafion in the stretching OH region (2500-4000 cm -1). Our findings indicated that the secondary structure of water species in Nafion can be accurately explained in terms of our reactive force field for water. The distinction between "surface" and "bulk" water contributions in Nafion membrane pores is proposed along with a quantitative estimate of the different types of OH groups present in the system. The average pore size was calculated and supported by the spectral results. © 2009 American Chemical Society.