The effects of hydraulic retention time (HRT) and substrate concentration on a two-chambered microbial fuel cell (MFC) were studied. The MFC was operated in a continuous flow mode employing enriched hydrogen-producing mixed bacteria as the anodic inoculum and artificial sucrose wastewater as a substrate. The results showed that when the substrate concentration was high, this system exhibited rapid start-up and continuous and stable power output over a long period. HRT and substrate concentration had a great effect on the electricity-generation characteristics of the MFC. The optimum HRT was 8 h, and the maximum power density was produced using a sucrose concentration of 3.5 g/L (HRT = 10 h). Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.