Abstract: Effects of different parameters including age of inoculum, size of inoculum and headspace on hydrogen production were investigated in detail using Rhodobacter sphaeroides NCIMB 8253 in batch culture. The effects of aerobic and anaerobic conditions for growth of this bacterium were also studied. The growth of R. sphaeroides NCIMB 8253 in aerobic condition reached exponential phase faster as compared to the anaerobic condition. The maximum absorbance at 660nm for the aerobic culture was 20% higher than the cells in anaerobic condition. The lag phase has been shortened when same medium was used for inoculum and H2 production but the H2 yield was found 9.26% lower as compared when different media were used for inoculum and H2 production. It was found that inoculum age 24 h, 10% v/v inoculum size with initial pH 7 can be designated as optimum conditions for maximum H2 production.