Rhodobacter capsulatus produces molecular hydrogen under the photoheterotrophic growth condition with reduced carbon sources (organic acids). Under this condition, ubiquinol pool is over reduced and excess reducing equivalents are primarily consumed via the reduction of CO2 through the Calvin-Benson-Bassham (CBS) pathway, the dimethylsulfoxide reductase (DMSOR) system or by the reduction of protons into hydrogen gas with the use of nitrogenase to maintain a balanced intracellular oxidation-reduction potential (redox balance). In order to investigate the effect of redox balancing pathways on nitrogenase-dependent hydrogen production, CO2 fixation was blocked by inactivating the phosphoribulokinase (PRK) of CBS pathway in wild type (MT1131), uptake-hydrogenase deficient strain (YO3), and cyt cbb(3) oxidase and uptake-hydrogenase deficient double mutant (YO4) strains. The hydrogen production properties of newly generated strains deficient in the CBB pathway were analyzed and compared with wild type strains. The obtained data indicated that, the total hydrogen production was increased slightly in CBB deficient mutant of YO3 and YO4 (4.7% and 12.5% respectively). Moreover, the maximum hydrogen production rate was increased by 13.3% and 12.7% for CBB deficient mutant of MT1131 and YO3 respectively. It was also observed that under the photoheterotrophic growth condition with ammonium as a nitrogen source, PRK deficient strains gave photoheterotrophically competent ammonium insensitive revertants. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.