We report on an alternative concept of biofuel cell functioning based on the unconventional use of enzymes to create a pH difference generating a potential difference between electrodes soaked in quinhydrone solutions. The electrode and quinhydrone solution were confined in a dialysis bag placed into a compartment containing either glucose oxidase and catalase for the biocathode or urease for the bioanode. In presence of 0.4 mol L-1 glucose and urea, the enzyme reactions generate a pH difference of 3.55, both compartments being separated by an agar-agar wall. The resulting biofuel cell exhibits an open-circuit voltage and maximum power of 208 mV and 30.6 mu W, respectively, without immobilization and electrical connection of the involved enzymes. In addition, this biofuel cell was able to provide continuously 10 mu A during 23 h, producing 0.133 J and 0.828 C. A similar biofuel cell configuration based only on dialysis bags was also developed. A graphite disk electrode elaborated by mechanical compression of graphite particles and quinhydrone, was placed in a dialysis bag itself confined into another dialysis bag containing enzyme solution. The resulting power and open-circuit voltage at saturating substrate conditions are 7.6 mu W and 157 mV, respectively. (C) 2010 Elsevier B.V. All rights reserved.