The electron transfer mechanism in enzymatic electrodes employing tetrathiafulvalene-tetracyanoquinodimethane (TTF-TCNQ) complex is despite of numerous publications, still a matter of controversy. To clarify this issue, enzymatic electrodes based on TTF, TCNQ and TTF-TCNQ have been prepared in an identical manner and their electrochemical behavior and activity have been tested. The enzymatic electrodes containing the respective mediator, glucose oxidase and Vulcan nanoparticles dispersed in a gelatin matrix show high activity for glucose oxidation in terms of both currents and oxidation onset potential. The observed electrochemical features, supported by infrared spectroscopy measurements, indicate that the activity of the TTF-TCNQ electrodes can be ascribed to TTF and TCNQ species released from the organic salt. The two mediators are active in different potential regions, implying an alternating electron transfer mechanism. TTF is active at more negative potentials and generates higher current densities, while TCNQ exhibits activity only at potentials more positive than 0 V vs. SCE. This work analyzes and summarizes different aspects of the most recent electron transfer hypotheses by providing the respective experimental evidences in an effort for better understanding of this puzzling bioelectrochemical system. (C) 2012 Elsevier B.V. All rights reserved.