The structure of human carbonic anhydrase II at pH 9.5 has been studied by X-ray crystallographic methods to 2.2 A resolution. These studies complement those performed under acidic conditions in which the catalytically-important proton-shuttle group, His-64, exhibits conformational mobility about side-chain torsion angle chi 1. However, no structural changes are observed in the conformation of His-64 at high pH. Therefore, we conclude that the protonation of His-64 (as well as zinc-bound hydroxide) may be a factor which contributes to the predominantly "out" conformation for His-64 observed at low pH.