Cooperative oxygen binding by vertebrate tetrameric hemoglobins (Hbs) has been extensively studied and is relatively well understood. Nonetheless, Hill coefficients greater than four have been reported for adult avian, amphibian, and reptilian red blood cells. Such reports also exist for embryonic red cells from various animals. These results are controversial and not yet convincingly established. Oxygen binding studies on avian Hb D, which is known to undergo deoxygenation-dependent self-association, were carried out to answer this question. The goal was to determine unequivocally whether Hill coefficients greater than four occur. Such high Hill coefficients were observed but only at very high Hb D concentrations. Moreover, the early model of avian deoxy Hb D self-association was found to be incomplete. The model has now been expanded to describe better the observed sedimentation data at high Hb concentrations. The possibility that embryonic deoxy Hbs self-associate was also assessed by sedimentation studies of deoxygenated Hb solutions from a marsupial, the tammar wallaby. The results obtained show unambiguously that these embryonic Hbs self-associate upon deoxygenation. Recent phylogenetic analyses suggest that the avian [alpha superscript D]-globin originated from embryonic [alpha]-globins. This finding suggests that the propensity to self-associate upon deoxygenation is an intrinsic property of tetrameric Hbs with embryonic [alpha]-globins. Furthermore the residues mediating the inter-tetramer interactions in adult avian deoxy Hb D and embryonic deoxy Hbs are likely to be the same. Recombinant globins were expressed in bacteria and protocols for the assembly of avian recombinant tetrameric Hb D developed. Initial measurements by sedimentation were carried out to verify the role of a conserved glutamate residue previously speculated to be involved in inter-tetramer interactions. The present studies provide a framework for future investigations of deoxygenation-dependent Hb self-association. In particular the need to carry out oxygen equilibrium measurements at high Hb concentrations as well as sedimentation studies of the deoxygenated Hb solutions is stressed. / text