The binding mode between oxyhemoglobin (oxyHb) and adenosine triphosphate (ATP) has been studied using absorption and fluorescence spectroscopy. OxyHb forms a ground state complex with ATP supported by five isosbestic points which appear in absorption spectra of oxyHb in buffer solution on addition of ATP. Moreover, the changes in absorption spectra suggest an oxidative interaction between the particular interacting systems. The binding constant has been determined from the quenching of fluorescence of oxyHb in the presence of a varied concentration of ATP, and that is 3.8 x 10(3) M-1 at 25 degrees C. The negative changes in entropy and enthalpy indicate that the binding is enthalpy driven and the hydrogen bond and van der Waals (stacking) interactions play a major role. The oxygen affinity of oxyHb decreases with simultaneous formation of metHb in the presence of ATP. ATP-induced structural changes have been affirmed using both circular dichroism spectroscopy and synchronous fluorescence. A theoretical docking study gives the molecular details about the binding site of ATP in oxyHb.