The use of glycerol for hydrogen gas production was examined via electrohydrogenesis using microbial electrolysis cells (MECs). A hydrogen yield of 3.9 mol-H(2)/mol was obtained using glycerol, which is higher than that possible by fermentation, at relatively high rates of 2.0 +/- 0.4 m(3)/m(3) d (E(ap) = 0.9 V). Under the same conditions, hydrogen was produced from glucose at a yield of 7.2 mol-H(2)/mol and a rate of 1.9 +/- 0.3 m(3)/m(3) d. Glycerol was completely removed within 6 h, with 56% of the electrons in intermediates (primarily 1,3-propanediol), with the balance converted to current, intracellular storage products or biomass. Glucose was removed within 5 h, but intermediates (mainly propionate) accounted for only 19% of the electrons. Hydrogen was also produced using the glycerol byproduct of biodiesel fuel production at a rate of 0.41 +/- 0.1 m(3)/m(3) d. These results demonstrate that electrohydrogenesis is an effective method for producing hydrogen from either pure glycerol or glycerol byproducts of biodiesel fuel production. (C) 2009 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.