This study reports the use of cyclic voltammetry (CV), hydrodynamic voltammetry, and electrochemical impedance spectroscopy (EIS) to investigate horseradish peroxidase (HRP) direct adsorption onto a glassy carbon (GC) surface and the adsorption of HRP in the presence of polymyxin (PM) forming Nafion (R)-covered HRP-PM films on a GC surface. The bioelectrocatalytic behavior of these electrodes toward H2O2 and O-2 reduction was also studied. The electrochemical reaction rate constant of HRP-PM/Nafion films was comparable to that of bare GC electrodes containing HRP and carbon nanotubes or HRP and graphene in their films. GC/HRP-PM/Nafion electrodes were sufficiently bioelectrocatalytic for H2O2 and O-2 reduction (even at pH 7 and 37 degrees C)-a feature that suggests their possible use as negative electrodes in biofuel cells. From EIS experiments, it was possible to estimate the resistance to electron hopping (R-eh) between heme Fe3+/Fe2+ redox centers in HRP enzymes and between these and H2O2 or O-2 molecules. (C) 2012 Elsevier Ltd. All rights reserved.