We have defined the predominant site of p-nitrophenyl acetate reaction with hemoglobin. The site is involved with, at least, two modes of action: the imidazole catalysis of His beta 2 and the irreversible covalent acetylation of Lys beta 82. The effect of competitive inhibition of the reaction by 2,3-diphosphoglyceric acid, the dependence of the reaction rate on the protein conformation, hemoglobin mutants, and the diethylpyrocarbonate are consistent with the assignment of the active site. In addition, the results point to small conformational differences in the NH-terminal regions of the beta chains between Hb S and Hb A.