It is generally believed that bovine hemoglobin (BvHb) interacts weakly with 2,3-diphosphoglycerate (2,3-DPG) in a chloride-free media and not at all in the presence of physiological concentrations of chloride (100 mM). This lack of interaction has raised several questions at both structural and evolutionary levels. Results obtained in this study via 31P nuclear magnetic resonance (NMR) show that, even in the presence of 100 mM chloride ions, 2,3-DPG does, in fact, interact with bovine deoxy-Hb. This spectroscopic observation has been confirmed by oxygen binding experiments, which have also shown that, under certain conditions, chloride and 2,3-DPG may display a synergistic effect in modifying the oxygen affinity of bovine hemoglobin. It could be that this synergistic effect has its structural basis in a conformational modification induced by 2,3-DPG, possibly causing extra chloride anions to approach the positive charges which constitute the anion binding site. Another possibility, not necessarily an alternative, is the additional chloride binding site recently identified [Fronticelli, C., Sanna, M. T., Perez-Alvarado, G. C., Karavitis, M., Lu, A.-L., and Brinnigar, W. S. (1995) J. Biol. Chem 270, 30588-30592] involving lysine beta76 that in bovine Hb substitutes for the alanine residue present in human hemoglobin. All of these findings are in agreement with the very low enthalpy of oxygenation that characterizes bovine Hb when both chloride and 2,3-DPG are present in concomitance. The results reported here clearly show that bovine hemoglobin does react with 2, 3-DPG and is functionally affected by this organic phosphate. Hence, the very low intraerythrocytic concentration of 2,3-DPG (0.5 mM) in adult bovine red blood cells is the result of metabolic adaptation which cannot be explained solely by the different amino acid sequence at the level of the 2,3-DPG binding site.