The active-site protonation state is crucial to the reductive mechanism of Escherichia coli thioredoxin, which involves a nucleophilic attack by the thiolate form of Cys32. We have calculated the titration properties of the active-site residues using a continuum electrostatic model, the X-ray structure of the oxidized protein, and ensembles of NMR structures of the oxidized and reduced protein. Protein dipoles, especially the SH dipole of Cys35, can provide sufficient stabilization of the Cys32 thiolate to account for its low experimental pKa (approximately 7.4), but this effect is very sensitive to local conformational variations. The experimental finding that Cys32 titrates at a lower pH than Cys35 is explained by the latter's deeper burial from solvent exposure, and stronger interaction with the carboxylate of Asp26, and not by helix dipoles or positively charged side chains. The calculated very strong interaction between Cys32 and Cys35 in their thiolate forms implies that their titration must occur in two widely pH-separated steps and that the thiolate groups must move apart in the second step. The calculations are very consistent with the experimental Asp26 pKa value of 7.5 for the oxidized X-ray structure. Both the oxidized and reduced NMR structures fall into two categories: "tight" structures in which the Asp26 and Lys57 side chains are in direct contact, and for which the calculations predict unreasonably low pKas; and "loose" structures, which resemble the oxidized X-ray structure in that these side chains are farther apart, and for which the calculations are in very good agreement with experiment. We propose that the calculations over the NMR ensemble can be used as a test of the alternative structural models provided by NMR.