The effects of mixed solvents on the ligand binding site in hemoglobin have been investigated though three spectroscopic techniques. Two classes of organic solvents (amides and alcohols) known to increase or decrease the hemoglobin affinity have been chosen for this study. The analysis of the iron CO stretching band shows that the ligand binding sites of alpha CO and beta CO subunits inside the alpha 2 beta 2 hemoglobin tetramer exhibit multiple conformations. From the circular dichroism and X-ray absorption near-edge structure data, it appears that no core deformation or heme reorientation occur with the affinity changes. The iron-ligand average bond angle is the sole parameter that depends on the external solvent. Since cosolvents seem to affect the dynamics rather than the hindrance of the heme cavity, we suggest that the protein affinity could be associated with a hierarchy of subtle dynamic states.