Several vital functions/physical characteristics of erythrocytes (including glycolysis, the pentose phosphate pathway, ion fluxes and cellular deformability) display dependence on the state of hemoglobin oxygenation. The molecular mechanism proposed involves an interaction between deoxyhemoglobin and the cytoplasmic domain of the anion exchange protein, band 3 (cdB3). Given that band 3 also binds to membrane proteins 4.1 and 4.2, several kinases, hemichromes and integral membrane proteins, and at least three glycolytic enzymes, it has been suggested that the cdB3-deoxyhemoglobin interaction might modulate the pathways mediated by these associated proteins in an oxygen-dependent manner. We have investigated this mechanism by synthesizing 10-mer peptides corresponding to the N-terminal fragments of various vertebrate cdB3s, determining their effects on the oxygenation reactions of hemoglobins from the same and different species and examining binding of the glycolytic enzyme glyceraldehyde-3P-dehydrogenase to the erythrocytic membrane of mouse erythrocytes. The cdB3 interaction is strongly dependent on pH and the number of negative and positive charges of the peptide and at the effector binding site, respectively. It lowers the oxygen association equilibrium constant of the deoxygenated (Tense) state of the hemoglobin and is inhibited by magnesium ions that neutralize cdB3's charge and by 2,3- diphosphoglycerate that competes for the cdB3-binding site. The interaction is stronger in humans (whose erythrocytes derive energy predominantly from glycolysis and exhibit higher buffering capacity) than in birds and ectothermic vertebrates (whose erythrocytes metabolize aerobically and are poorly buffered) and is insignificant in fish, suggesting that its role in the regulation red cell glycolysis increased with phylogenetic development in vertebrates.