In this study, the Gram-positive aerobic bacterium Bacillus subtilis has for the first time been employed in a microbial fuel cell (MFC). A glucose-fed MFC with M9 minimal medium in the anode chamber was operated for 3 months, establishing a highly active MFC using filtered M9 medium as the catholyte, carbon cloth as the anode and a 20% platinum electrode as the cathode. The bioelectrical responses of the MFC were characterized by the circuit potential, measured at an average value of 370 mV. A potential of 115 mV appeared to characterize the maximum power produced from a polarization test was 1.05 mW cm -2 at a resistance of 0.56 k . In situ cyclic voltammograms with and without biofilm anodes were performed in the growth phase and showed that redox metabolites were produced, which varied with physiological status. Voltammograms obtained from a comparative study of broth, supernatant and resuspended bacterial cells revealed that the electrochemical activity in the anode chamber arose from the redox compounds in the supernatant. The results show that the microorganism B. subtilis is electrochemically active and that the electron transfer mechanism is mainly due to the excreted redox compounds (mediator) in the broth solution and not to the membrane-bound proteins.