The α and β chains of human hemoglobin are functionally nonequivalent. In tetrameric human methemoglobin, the β chains react with sodium dithionite about 10-fold faster, and with sodium azide about 6-fold faster than the α chains. In addition, the isosbestic point for oxidized heme-reduced heme differs for the α and β chains within the intact tetramer. The differential reactivity of the chains toward azide and dithionite was used to measure the concentrations of the oxidized α and β chains in mixtures of methemoglobin and deoxyhemoglobin. The ratio of concentration of oxidized β to oxidized α chains decreased as the proportion of methemoglobin was progressively decreased, in the presence of catalytic amounts of the electron-mediating dye thionine. Therefore, the β chains have a higher half-reduction potential than the α chains. Furthermore, nearly the same difference in apparent oxidation potential was found at pH 6.1, where the Hill coefficient for the oxidation-reduction reaction is 1.4, and at pH 8.7, where the Hill coefficient is 2.5. We obtained an estimate of the heme-heme interaction energy contributed by the αβ dimer from these results and from the known equilibrium curves for the oxidation-reduction reaction. The estimated αβ dimer interaction energy is much smaller than the experimentally measured interaction energy of the tetramer. We conclude that since the αβ dimer contributed only a small fraction of the total interaction energy at alkaline pH, the hemoglobin tetramer is the important functional unit in the oxidation-reduction reaction.