H(2) can be produced from organic matter with a microbial electrolysis cell (MEC). To decrease MEC capital costs, a cathode is needed that is made of low-cost material and produces H(2) at high rate. A microbial biocathode is a low-cost candidate, but suffers from a long startup and a low H(2) production rate. In this study, the effects of cathode potential and carbon source on microbial biocathode startup were investigated. Application of a more negative cathode potential did not decrease the startup time of the biocathode. If acetate instead of bicarbonate was used as carbon source, the biocathode started up more than two times faster. The faster startup was likely caused by a higher biomass yield for acetate than for bicarbonate, which was supported by thermodynamic calculations. To increase the H(2) production rate, a flow through biocathode fed with acetate was investigated. This biocathode produced 2.2 m(3) H(2) m(-3) reactor day(-1) at a cathode potential of -0.7 V versus NHE, which was seven times that of a parallel flow biocathode of a previous study. Copyright © 2011 Wiley Periodicals, Inc.