The aim of this study was to investigate the development of cathodic biofilm and its effect on methane production in a single-chamber microbial electrolysis cell (MEC). The MEC with 1 g/L acetate was successfully operated within 31 cycles (similar to 2400 h). The maximum methane production rate and average current capture efficiency in the MEC reached 93 L/m(3).d and 82%, respectively. Distinct stratification of Methanobacteriaceae within cathodic biofilm was observed after 9 cycles of operation. The relative abundance of Methanobacteriaceae in the microbial community increased from 45.3% (0-15 mu m), 57.6% (15-30 mu m), 66.9% (30-45 mu m) to 77.2% (45-60 mu m) within the cathodic biofilm. The methane production rates were positively correlated with the mcrA gene copy numbers in the cathodic biofilm. Our results should be useful to understand the mechanism of methane and hydrogen production in the MEC.