The presence of microorganisms on cathodes has been shown to enhance the hydrogen evolution reaction (HER), but a requirement for viable cells has not been sufficiently examined. HER was examined using live or killed biocathodes of exoelectrogenic (Geobacter sulfurreducens) and non-exoelectrogenic (Escherichia coli) bacteria, and a hydrogenotrophic methanogen (Methanosarcina barkeri). Electrodes at a set potential of -0.6 V (versus a standard hydrogen electrode) containing G. sulfurreducens biofilms or killed controls produced hydrogen at a similar rates (118 ± 15 nmold-1 mL-1) over 5 months. Electrodes containing cell extracts produced hydrogen at approximately half this rate (56 ± 6 nmold-1 mL-1). Biocathodes fed lactate produced only 14 ± 2 nmol/d-mL. Electrodes inoculated with M. barkeri produced hydrogen at a rate (120 ± 18 nmold-1 mL-1) similar to the G. sulfurreducens, but no methane was recovered after the initial inoculation cycle. Non-exoelectrogenic E. coli cells and extracts produced hydrogen at a slower rate (13 ± 1 and 4 ± 1 nmold-1 mL-1, over 3 cycles). Electrodes exposed to viable cells that were examined after 5 months had increased levels of in nitrogen, sulfur, iron, nickel, cobalt, and peptides (possibly remnants of hydrogenases and other oxidoreductases) relative to uninoculated controls, and no intact cells. These results show that enhanced HER can result from cell debris and that living cells are not required.