The major quaternary structural change responsible for the allosteric transition in human adult hemoglobin (Hb) from low-affinity T- conformation to high-affinity R-conformation upon ligand binding to hemes has been described as a 15-degree rotation of one alpha1beta1 dimer over the pairing one. This interdimeric event has been in the limelight for decades. However, since very few changes, if any, have been reported for the alpha1beta1 intradimeric interface, its contribution to allostery has been neglected. Here, dramatic changes in allosteric characteristics that are caused by a specific steric alteration of the alpha1beta1 interface by chemical modification are reported.The chemically-modified alpha1beta1 interface Hb showed an extremely low affinity for oxygen and an almost complete absence of cooperativity, comparable to those caused in native Hb by a combination of strong allosteric effectors. The functional characterization is supported by structural evidence revealed by EPR using the nitrosyl derivative, which resembles that one characterized by an extremely low affinity for the ligand showing a triplet superhyperfine around g = 2. Sedimentation experiments showed that modified dimers associated tightly into tetramers. Calorimetric experiments, on the other hand, revealed that the stabilizing interactions were mainly hydrophobic.The above-mentioned findings suggest that the alpha1beta1 interface is not inert, but rather an extremely important interface playing a pivotal role on the control of oxygen affinity.