The intrinsic pKa values of protons of 2,3-diphosphoglycerate (DPG) which titrate in the physiologically significant range (i.e., pH 6.8-7.8) have been determined by measuring the changes in chemical shifts of the two phosphate resonances of the molecule as a function of pH using 31P-NMR spectroscopy. While conventional acid-base titration techniques resulted in apparent pKa values of 6.39 and 7.39 for these protons, analysis of the 31P-NMR data by statistical thermodynamic methods yielded intrinsic pKa values of 6.99 +/- 0.07 and 7.28 +/- 0.04, for protons associated with the phosphates bound to carbon-3 (C-3) and carbon-2 (C-2), respectively, with an interaction energy of +0.77 kcal/mol. The free energies for the binding of protons to the C-2 and C-3 phosphates and the associated interaction energies determined by 31P-NMR were used to generate a theoretical titration curve which was essentially identical to that determined by conventional acid-base titration. The physiological implications of this work are briefly discussed.