Oxygen equilibria were measured on a number of human hemoglobins, which had been “stripped” of organic phosphates and isolated by column chromatography. In the presence of 2 × 10^-4 M 2,3-diphosphoglycerate (2,3-DPG), the P₅₀ of hemoglobins A, A₂, S, and C increased about twofold, signifying a substantial and equal decrease in oxygen affinity. Furthermore, hemoglobins Chesapeake and M_Milwaukee-1 which have intrinsically high and low oxygen affinities, respectively, also showed a twofold increase in P₅₀ in the presence of 2 × 10^-4 M 2,3-DPG. In comparison to these, hemoglobins A_IC and F were less reactive with 2,3-DPG while hemoglobin F_I showed virtually no reactivity. The N-terminal amino of each β-chain of hemoglobin A_IC is linked to a hexose. In hemoglobin F_I the N-terminal amino of each γ-chain is acetylated. These results suggest that the N-terminal amino groups of the non-α-chains are involved in the binding of 2,3-DPG to hemoglobin.