Abstract: Hydrogen is considered as a promising alternative fuel and energy carrier by virtue of the fact that it does not evolve the CO2 in combustion. Some non-sulfur photosynthetic bacteria are potent hydrogen producers, utilizing organic acids or alcohols under specific environmental conditions. Effects of different parameters including different initial pH, use of argon and nitrogen gas on hydrogen production were investigated in detail using R. sphaeroides NCIMB 8253 in batch culture. The cultures were grown aerobically and anaerobically and hydrogen production was investigated at different initial pH ranging from 6-11. It was found that initial pH 7 can be designated as optimum pH for maximum cumulative H2 production of 45.03 mL, yield per gram of substrate (225.17 mL g-1) and rate of H2 production (3.13 mL/L/h) compared to other initial pH within the range of 6-11. Argon gas was found better than nitrogen gas to create more suitable anaerobic conditions for maximum H2 production using R. sphaeroides NCIMB 8253. Culture sparged with argon gas at the beginning produced 45.03 mL H2 after 144 h incubation; whereas culture sparged with N2 gas only produced 23.50 mL H2 after 120 h incubation. The findings reveal that the use of argon gas to create anaerobic environment and the initial pH 7 could favor the enhanced hydrogen production using Rhodobacter sphaeroides NCIMB 8253.