In this paper hydrogen production through biocatalyzed electrolysis was studied for the first time in a single chamber configuration. Single chamber biocatalyzed electrolysis was tested in two configurations: (i) with a cation exchange membrane (CEM) and (ii) with an anion exchange membrane (AEM). Both configurations performed comparably and produced over 0.3 m3 H2/m3 reactor liquid volume/day at 1.0 V applied voltage (overall hydrogen efficiencies around 23%). Analysis of the water that permeated through the membrane revealed that a large part of potential losses in the system were associated with a pH gradient across the membrane (CEM DeltapH=6.4; AEM DeltapH=4.4). These pH gradient associated potential losses were lower in the AEM configuration (CEM 0.38 V; AEM 0.26 V) as a result of its alternative ion transport properties. This benefit of the AEM, however, was counteracted by the higher cathode overpotentials occurring in the AEM configuration (CEM 0.12 V at 2.39 A/m2; AEM 0.27 V at 2.15 A/m2) as a result of a less effective electroless plating method for the AEM membrane electrode assembly (MEA).