The effects of 4-aminoantipyrine (AAP) on bovine hemoglobin (BHb) were investigated by fluorescence spectroscopy, synchronous fluorescence spectroscopy, ultraviolet-visible absorption spectroscopy and circular dichroism spectroscopy (CD) under simulated physiological conditions. The experimental results showed that AAP effectively quenched the intrinsic fluorescence of BHb via static quenching. The number of binding sites, the binding constant K(a), and the thermodynamic parameters (DeltaH(o), DeltaS(o) and DeltaG(o)) were measured at two different temperatures. Van der Waals' interactions and hydrogen bonds were the predominant intermolecular forces in stabilizing the BHb-AAP complex. The experiment results confirmed micro-environmental and conformational changes of BHb in the presence of AAP. The alpha-helix content decreased, indicating that AAP destroys some of the hydrogen bonding networks in the polypeptide chain.