The attenuated total reflection/Fourier transform infrared technique has been utilized to characterize secondary structural changes in human serum albumin (HSA) before and after protein binding via incubation of HSA in different concentrations of ethanol, captopril or ethanol/captopril mixture. The results indicate that ethanol induced a transition from beta-sheet to an alpha-helical structure and promoted conversion of intramolecular hydrogen-bonded beta-sheet to intermolecular hydrogen-bonded beta-sheet. In contrast, captopril or captopril/ethanol mixture induced conversion of intramolecular hydrogen-bonded beta-sheet to intermolecular hydrogen-bonded beta-sheet and resulted in exposure of the aromatic side-chain groups in the unfolding conformation of HSA. Thus, protein binding between HSA and captopril or captopril/ethanol seems to play an important role in protein secondary structure.