A new recombinant, human anti-sickling β-globin polypeptide designated βAS3 (βGly16 → Asp/βGlu22 → Ala/βThr87 → Gln) was designed to increase affinity for α-globin. The amino acid substitutions at β22 and β87 are located at axial and lateral contacts of the sickle hemoglobin (HbS) polymers and strongly inhibit deoxy-HbS polymerization. The β16 substitution confers the recombinant β-globin subunit (βAS3) with a competitive advantage over βS for interaction with the a-globin polypeptide. Transgenic mouse lines that synthesize high levels of HbAS3 (α2βAS32) were established, and recombinant HbAS3 was purified from hemolysates and then characterized. HbAS3 binds oxygen cooperatively and has an oxygen affinity that is comparable with fetal hemoglobin. Delay time experiments demonstrate that HbAS3 is a potent inhibitor of HbS polymerization. Subunit competition studies confirm that βAS3 has a distinct advantage over βS for dimerization with α-globin. When equal amounts of βS- and βAS3-globin monomers compete for limiting α-globin chains up to 82% of the tetramers formed is HbAS3. Knock-out transgenic mice that express exclusively human HbAS3 were produced. When these mice were bred with knock-out transgenic sickle mice the βAS3 polypeptides corrected all hematological parameters and organ pathology associated with the disease. Expression of βAS3-globin should effectively lower the concentration of HbS in erythrocytes of patients with sickle cell disease, especially in the 39% percent of these individuals who coinherit α-thalassemia. Therefore, constructs expressing the βAS3- globin gene may be suitable for future clinical trials for sickle cell disease.