A combined experimental design and molecular modelling study of the efficiency of hematin/HRP-H2O2 in Alizarin Red S (ARS) removal from aqueous solutions is reported. A Doehlert experimental design using 21 experiments, with temperature range fixed to 30-50 degrees C, pH range 7-11, hydrogen peroxide concentration from 0.05 to 5 mM and catalyst concentration from 0.05 to 4.95 mg/l is used. The factor levels are 7 for H2O2 concentration and pH, 5 for temperature and 3 for catalyst concentration. At optimal conditions, 75 mg/l ARS solutions are totally decolorized by hematin whereas HRP only achieves 57% conversion. UV/visible spectra analysis of reaction medium, species profiles during reaction using HRP/hematin at selected conditions and molecular modelling using molecular mechanics (MM2) and semiempirical studies (PM6-MOPAC 2009) are presented and discussed to explain the experimental findings. Cpd-I of HRP coordinates selectively ARS vs. hydrogen peroxide. Dimers of ARS may inhibit HRP. ARS may be coordinated by different hydroxyls to hematin depending on the availability of them, considering the pH. Catalatic reaction is important in the case of hematin but it is not in the case of HRP in our range of experimental conditions. (C) 2011 Elsevier B.V. All rights reserved.