The effect of allosteric effectors, such as inositol hexakisphosphate and/or bezafibrate, has been investigated on the unliganded human adult hemoglobin both spectroscopically (employing electronic absorption, circular dichroism, resonance Raman, and x-ray absorption near-edge spectroscopies) and functionally (following the kinetics of the first CO binding step up to a final 4% ligand saturation degree). All data indicate that the unliganded T-state is not perturbed by the interaction with either one or both effectors, suggesting that their functional influence is only exerted when a ligand molecule is bound to the heme. This is confirmed by the observation that CO dissociation from partially liganded hemoglobin ( </= 0.04) is strongly altered by the presence of either effector, and the effect is enhanced whenever the two effectors are simultaneously present. Altogether, these data are a direct demonstration of the occurrence of a strain induced by the presence of a ligand molecule bound to the heme, and for the first time there is a clear indication that the expression of the functional heterotropic effect by these non-heme ligands requires this strain, which is not present in the unliganded molecule.