The mechanism and thermodynamic parameters for the binding of luminol (LH2) with human serum albumin was explored by steady state and picosecond time-resolved fluorescence spectroscopy. It was shown that out of two possible LH2 conformers present is solution, only one is accessible for binding with HSA. The thermodynamic parameters like enthalpy (Delta H) and entropy (Delta S) change corresponding to the ligand binding process were also estimated by performing the experiment at different temperatures. The ligand replacement experiment with bilirubin confirms that LH2 binds into the sub-domain IIA of the protein. (C) 2010 Elsevier B. V. All rights reserved.