The reaction of a soluble thiamine-binding protein of Saccharomyces cerevisiae with water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, at pH 4.5, results in a remarkable loss of its binding activity with thiamine. Thiamine above 0.1 mM substantially protects the protein against this inactivation. In addition to 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, the thiamine-binding protein is also inactivated by diethylpyrocarbonate. The inactivation is time-dependent and follows second-order kinetics. Restoration of the binding activity by incubation of inactivated protein with hydroxylamine was observed. thiamine and pyrithiamine are effective to prevent the inactivation. From these results it is strongly suggested that both the carboxyl and the histidine residues in the protein are involved in the binding site for thiamine. It is proposed that the binding involves interactions between charged groups on the protein with the quaternary nitrogen of the thiazolium moiety and with the basic ring nitrogen of the pyrimidine moiety in thiamine molecule.