Transgenic swine expressing human HbA contained only one of two types of the anticipated interspecies hybrids, namely H alpha 2 P beta 2 (H = human, P = swine). In an attempt to establish whether the absence of the swine alpha and human beta (P alpha 2 H beta 2) hybrid in vivo is a reflection of the lack of complementarity between the interspecies chains to generate appropriate interfaces, we have undertaken the in vitro assembly of swine alpha and human beta chimeric tetramer. In contrast to the in vivo transgenic swine system, in vitro the hybrid of swine alpha human beta chain is assembled readily and the hybrid exhibits normal cooperative oxygen binding. Both the swine alpha human beta and the human alpha swine beta interspecies hybrids are stable around neutral pH and do not segregate into parent tetramers even when mixed together. On the other hand, nearly complete exchange of P alpha chain of P alpha 2 H beta 2 hybrid occurs in the presence of H alpha chain at pH 6.0 and room temperature, resulting in the formation of HbA. However, very little of such an exchange reaction takes place at pH 7.0. These results suggest that the thermodynamic stability of P alpha 2 H beta 2 hybrid is lower compared to that of HbA. In contrast, P beta chain of H alpha 2 P beta 2 hybrid is refractory to exchange with H beta chain at pH 7.0 as well as at pH 6.0, suggesting that the stability of H alpha 2 P beta 2 is higher compared to that of HbA (H alpha 2 H beta 2). The swine alpha human beta chimeric Hb undergoes subunit exchange reaction with human alpha-chain in the presence of 0.9 M MgCl2, at pH 7.0. This demonstrates the lower thermodynamic stability of the intradimeric interactions of the heterodimer even at neutral pH. A synergistic coupling of the intra- and interdimeric interactions of the swine alpha and human beta chain heterodimer is essential for the thermodynamic stability of the chimeric Hb under the physiological conditions. Accordingly, we speculate that the lower thermodynamic stability of P alpha H beta heterodimer (compared to the homodimers H alpha H beta and P alpha P beta) facilitates its segregation into the homodimers by subunit exchange reaction involving either H alpha or P beta. This molecular aspect by itself or possibly along with other cellular aspects of the swine system results in the absence of P alpha 2 H beta 2 hybrid in transgenic swine expressing HbA.