Cyclic voltammetry is an efficient means of analyzing the catalytic reduction of H2O2 at immobilized horseradish peroxidase (HRP)-Eastinan AQ 55 electrodes in the presence of 1,1 '-ferrocenedimethanol as a one-electron reversible cosubstrate. This system was employed to study the kinetics of the reduction of compound II of HRP in a number of organic solvents. An electrocatalytic response was detected in methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, acetone, 2-butanone, 1,2-propanediol, acetonitrile, ethyl acetate, and ethylene glycol. Unusual bell-shaped variations of the peak or plateau catalytic current with the substrate concentration were observed in all solvents tested. The results obtained in methanol, acetonitrile, and 1-propanol were analyzed using the model developed by Saveant (Limoges, B.; Saveant, J.-M.; Yazidi, D. J. Am. Chem. Soc. 2003, 125, 9192-9203). The values of h(3)Gamma(0) and K-3,K-M, where k(3) = k(3,1)k(3,2)/(k(3,-1) + k(3,2)), Gamma(0) is the surface concentration of active enzyme, and K-3,K-M = (h(3,-1) + k(3,2))/k(3,1), were determined. The values of k(3)Gamma(0) for the mediated reduction of compound II of HRP in methanol, 1-propanol, and acetonitrile (in the presence of 5% aqueous buffer) were not affected by the solvent dielectric constant but decreased with solvent hydrophobicity. The value of K-3,K-M obtained in methanol was similar to that obtained for [Os-(bPY)(2)pyCl](2+) in aqueous buffer.