A mass spectrometric method was developed to determine pH-dependent hydrogen-deuterium exchange at the C-2 position of the imidazole ring of histidine, after converting the amino acid to the methylthiohydantoin derivative. The amount of deuterium exchange in N-acetyl-histidine estimated by the present method was confirmed to be in good agreement with that determined by NMR spectrometry. N-Acetylhistidine was deuterated at various pH's. From the amount of deuterium exchange, a pseudo-first order rate constant (kθ) was calculated. A pka, value of 7.2 for the amino acid was obtained from the relation between kθ and pH. This method was applied to estimate the pka value of β-146 histidine in human hemoglobin. Human hemoglobin deuterated at various pH's was digested with carboxypeptidase A [EC 3.4.12.2] to release the β-146 histidine. The amount of deuterium exchange in the isolated histidine was determined to obtain kθ. From these measurements pka values of 7.0 for the histidine in oxyhemoglobin and of 8.2 for that in deoxyhemoglobin were found at 36.5°, respectively.