Single-walled carbon nanotubes (SWNTs) were coated first with methylene blue (MB) by noncovalent adsorption and then by horseradish peroxidase (HRP) by cross-linking with glutaraldehyde. The MB-SWNT/HRP composites formed stable films on glassy carbon electrodes. MB was probably present with a coverage of a monolayer or less. The MB voltammetry was consistent with fast electron transfer to a surface-confined species. The presence of HRP did not significantly affect the MB electrochemistry. MB could mediate electron transfer from HRP in the presence of H2O2. Cyclic voltammograms of this process were used to determine the rate constants for the reactions of the native ferriperoxidase with H2O2 and of the oxyferryl Compound II with the reduced form of MB. For comparison purposes, the rate constant for the direct electrode reduction of the HRP oxyferryl pi-cation radical Compound I was determined in MB-free SWNTs. The results indicate a considerably faster regeneration rate for native ferriperoxidase by the mediated reaction than by direct electrochemistry. Using the MB-SWNT/HRP composites, H2O2 could be calibrated by amperometry at -0.3 V vs. SCE. The optimized response (at pH 7.0) had a sensitivity of 661.0 mu A mM(-1) cm(-2) and a limit of detection (3 x S/N) of 0.1 mu M. (C) 2010 Elsevier Ltd. All rights reserved.