AbstractMetabolic control analysis (MCA) is an analytical technique that aims to quantify the distribution of control that enzymes exhibit over the steady‐state fluxes through a metabolic network. In an enzymatic biofuel cell, the flux of interest is the electrical current generated by the system. Regardless of transport limitations and other constraints, kinetic limitations can become potential bottlenecks in the operation of a biofuel cell. We have used an indirect approach to MCA to investigate a common osmium‐mediated glucose oxidase/laccase enzymatic biofuel cell. The results of the analysis show that the control of the electron flux strongly depends on the total mediator concentrations and the extent of polarization of the individual electrodes. The effect of varying oxygen concentrations is also examined, as oxygen is required for the cathode, but it participates in a non‐productive reaction at the anode. Under normal operating conditions the electrodes will be highly polarized and will both contain high mediator concentrations. This configuration will result in a dominant FCC at the anode, and the conditions that are needed for balanced flux control between the anode and cathode are explored. As increasingly complex bioelectrocatalytic systems and architectures are envisioned, MCA will be a valuable framework to facilitate their design and subsequent operation. Biotechnol. Bioeng. 2009;102: 1624–1635. © 2008 Wiley Periodicals, Inc.