Mitochondria modified electrodes have been developed and characterized that utilize whole mitochondria isolated from tubers and immobilized within a quaternary ammonium modified Nafion membrame on a carbon electrode that can oxidize pyruvate and fatty acids. Detailed characterization of the performance of these mitochondria modified electrodes has been accomplished by coupling the mitochondria-based bioanode with a commercial air breathing cathode in a complete pyruvate/air biofuel cell. The studies included the effect of fuel (pyruvate) concentration, mitochondria lysing, temperature and pH on the performance of the mitochondria catalyzed, pyruvate/air biofuel cell. Effect of oxygen and cytochrome c oxidase inhibitors on biofuel cell performance has allowed us to further understand the mechanism of electron transfer with the carbon electrode. (C) 2009 Elsevier Ltd. All rights reserved.