Alizarin Red S CARS), is a water-soluble, widely used anthraquinone dye synthesized by sulfonation of alizarin. In this report, the binding of ARS to human serum albumin (HSA) was characterized by employing fluorescence, UV/vis absorption, circular dichroism (CD), and molecular modeling methods. The data of fluorescence spectra displayed that the binding of ARS to HSA is the formation of HSA-ARS complex at 1:1 stoichiometric proportion. Hydrophobic probe 8-anilino-1-naphthalenesulfonic acid (ANS) was employed and elucidated that the dye was located in subdomain IIIA. This phenomenon corroborates the result of site-specific probe displacement experiments, which demonstrate the dye is at indole-benzodiazepine site (Sudlow's site II); and it is also consistent with guanidine hydrochloride (GuHCl) induced HSA unfolding studies and molecular modeling simulations. The features of the dye, which led to structural perturbations of HSA, have also been studied in detail by methods of UV/vis. CD and three-dimensional fluorescence spectroscopy. (C) 2010 Elsevier B.V. All rights reserved.