A cyclic voltammetric simulation that can be applied to an electrochemically mediated enzyme reaction involving any substrate and mediator concentrations was developed. Concentration polarization of the substrate in the vicinity of an electrode was considered as well as mediator concentration. Reversible and quasi-reversible electrochemical reactions with one electron followed by an enzyme reaction with two electrons were modeled. The differential equations for the mediator and substrate were solved using digital simulation techniques. The calculated cyclic voltammograms showed prepeaks when there was a low substrate concentration, high mediator concentration, and high enzyme activity. Digital simulation was applied to the determination of the kinetic constants of glucose oxidase (GOx). Cyclic voltammetry was carried out experimentally in a phosphate buffer solution containing GOx, ferrocene derivatives, and glucose. The ratio of the catalytic to the diffusion-controlled current, ik/id, was evaluated. The kcat, KMM, and KMS values were determined from the current values obtained by simulation and by experimentation at various enzyme, mediator, and substrate concentrations. The kcat, KMM, and KMS values for GOx, ferrocenedimethanol, and glucose were 340 s-1, 110 microM, and 30 mM, respectively.