Studies of chain folding in amylose employing spectroscopic techniques have led to an assignment of the 1295-cm−1band to a unique fold conformation. Irregular folds, present in single crystals, are regularized to the unique fold conformation during annealing. Annealing of solution-cast films, which initially contain a small amount of regular folding, increases the amount of regular folds. Swelling of these annealed films with water vapor destroys this unique folded conformation.