Effects of annealing have been studied on the chemical structure, water uptake, and acidity of the cast Nafion thin film of thickness similar to 6 mu m using a fluorescent probe, 2-(3'-pyridyl)benzimidazole (3PBI), and attenuated total reflactance infrared (ATR-IR) spectroscopy. Nonannealed films and thick Nafion 117 membrane have been used as reference materials, in order to develop a complete understanding of the effect. Annealing has been found to cause a decrease in acidity of otherwise highly acidic ionomer, as sensed by the fluorescent probe and loss of water, as reflected in the ATR-IR spectrum. This observation is surprising and cannot be explained in the light of previous reports of physical changes. Our ATR-IR study has revealed changes in the chemical structure of the hydrophilic part of the ionomer, leading to the formation of sulfonic acid anhydrides. This phenomenon can rationalize the decreased acidity reported in our fluorescence study. Interestingly, acid treatment of the annealed film restores the acidity of the unannealed films. This cannot be rationalized simply by a greater proton uptake from the solution, as the film has to be electroneutral. It appears that the anhydrides formed during the annealing process undergo acid hydrolysis, leading to an increase in the number of SO3 groups and, consequently, an increase in the number of H3O+ ions in the water channels. Besides, the films can be hydrated to an extent that is much greater than Nafion membranes, but the water uptake of acid treated annealed film is slightly less than that of nonannealed films. Hence, we conclude that, along with annealing, acid treatment, a procedure that is generally not performed on the cast films, is an important pretreatment procedure to improve the acidity and hence the transport properties of the cast film.