Potentiometric and amperometric measurements were made with microbial fuel cells containing E. coli or yeast as the anodic reducing agent and glucose as the oxidizable substrate. The catalytic effects of thionine and resorufin on the anode reaction were investigated. Results on the potentiometry, polarization, and coulombic output of the cells support a mediator‐coupled mechanism for the transfer of electrons from the organism to the electrode in preference to a mechanism of “direct” electrochemical oxidation of glucose or its degradation products. Experiments with ¹⁴C‐labeled glucose show that when a microbial fuel cell produces a current under load, exogenous glucose is metabolized to produce ¹⁴CO₂. The Coulombic yields of the cells indicate a high degree of energy conversion in these systems.