Ammonia inhibition of biohydrogen production was investigated in batch and continuous flow reactors with glucose as a substrate. In batch tests, biohydrogen production rate was highly dependent on pH and ammonia (defined as the sum of NH3 of NH4+ species) concentrations above 2 g N/L. At pH = 6.2, the maximum production decreased from 56 mL/h at 2 g N/L to 16 mL/h at 10 g N/L. At pH = 5.2, production decreased from 49 mL/h (2g N/L) to 7 mL/h (16 g N/L). Hydrogen yield remained relatively constant in batch tests, varying from 0.96 to 1.17 mol-H2/mol-glucose. In continuous flow tests, both hydrogen production rates and yields were adversely affected by ammonia. When the reactor (2.0 L) was first acclimated under batch conditions to a low nitrogen concentration (<0.8 g N/L), H2 production and yields under continuous flow mode conditions were 170 mL/h and 1.9 mol-H2/mol-glucose, but decreased with increased ammonia concentrations up to 7.8 g N/L to 105 mL/h and 1.1 mol-H2/mol-glucose. There was no hydrogen production under continuous flow conditions if the reactor was initially operated under batch flow conditions at ammonia concentrations above 0.8 g N/L. It is concluded that the hydrogen production is possible at high concentrations (up to 7.8 g N/L) of ammonia in continuous flow systems as long as the reactor is initially acclimated to a lower ammonia concentration (<0.8 g N/L).