The minor tetrameric hemoglobin (Hb), Hb D, of chicken red blood cells self-associates upon deoxygenation. This self-association enhances the cooperativity of oxygen binding. The maximal Hill coefficient is greater than 4 at high Hb concentrations. Previous measurements at low Hb concentrations were consistent with a monomer-to-dimer equilibrium and an association constant of ∼1.3–1.6 × 104M−1. Here, the Hb tetramer is considered as the monomer. However, new results indicate that the association extends beyond the dimer. We show by combination of Hb oligomer modeling and sedimentation velocity analyses that the data can be well described by an indefinite noncooperative or isodesmic association model. In this model, the deoxy Hb D associates noncooperatively to give a linear oligomeric chain with an equilibrium association constant of 1.42 × 104M−1 at 20°C for each step. The data are also well described by a monomer–dimer–tetramer equilibrium model with monomer-to-dimer and dimer-to-tetramer association constants of 1.87 and 1.03 × 104M−1 at 20°C, respectively. A hybrid recombinant Hb D was prepared with recombinant αD-globin and native β-globin to give a Hb D tetramer (αβ2). This rHb D undergoes decreased deoxygenation-dependent self-association compared with the native Hb D. Residue glutamate 138 has previously been proposed to influence intertetramer interactions. Our results with recombinant Hb D show that Glu138 plays no role in deoxy Hb D intertetramer interactions. Proteins 2011. © 2011 Wiley-Liss, Inc.