Abstract The role of selected amino acid residues in the monomer-monomer contacts of Hb A has been studied by site-directed mutagenesis of the α chain bearing substitutions in the subunit surfaces. Mutation α 38Thr → Trp induced a stabilization of tetrameric Hb-CO with a decrease of the K d for the equilibrium α 2β 2 ⇌ 2αβ, but had no effect on ligand binding. Mutation α40Thr→Arg resulted in a complete loss of cooperativity in ligand binding. Mutation α103His→Val had no noticeable effect. We also studied the behaviour of isolated, mutated α chains with respect to self association: compared to wt α chains, mutant α38Thr→Trp showed stabilization of the dimeric state and (at high protein concentration) a detectable amount of tetramers. Mutant α103His→Val showed only a minor stabilization of the α 2 dimer.