In order to assess the role of beta112 Cys in homo- and hetero-tetrameric hemoglobin formation, we expressed four beta112 variants (beta112Cys-->Asp, beta112Cys-->Ser, beta112Cys-->Thr, and beta112Cys-->Val) and studied assembly with alpha chains in vitro. beta112 Cys is normally present at beta1 beta2 and alpha1 beta1 interaction sites in homo- (beta4) and hetero-tetramers (alpha2 beta2). beta4 formation in vitro was influenced by the amino acid at beta112. beta112 Asp completely inhibited formation of homo-tetramers, whereas beta112 Ser showed only slight inhibition. In contrast, beta112 Thr or Val enhanced homo-tetramer formation compared with betaA chains. Association constants for homo-tetramer formation increased in the order of beta112Cys-->Ser, betaA, beta112Cys-->Thr, and beta112Cys-->Val, whereas the value for beta112Cys-->Asp was zero under the same conditions. These beta112 changes also affected in vitro alpha2 beta2 hetero-tetramer formation. Order of alpha2 beta2 formation under limiting alpha-globin chain conditions showed Hb betaC112S > Hb A > Hb S = Hb betaC112T = Hb betaC112V >>> Hb betaC112D. Hb beta112D can form tetrameric hemoglobin, but this beta112 change promotes dissociation into alpha and beta chains instead of alpha beta dimer formation upon dilution. These results indicate that amino acids at alpha1 beta1 interaction sites such as beta112 on the G helix play a key role in stable alpha beta dimer formation. Our findings suggest, in addition to electrostatic interaction between alpha and beta chains, that dissociation of beta4 homo-tetramers to monomers and hydrophobic interactions of the beta112 amino acid with alpha chains governs stable alpha1 beta1 interactions, which then results in formation of functional hemoglobin tetramers. Information gained from these studies should increase our understanding of the mechanism of assembly of multi-subunit proteins.