A series of batch tests were conducted to investigate the effects of pH and intermediate products on biological hydrogen production. The tests were run in serum bottles to determine the optimal operating conditions to maximize hydrogen production using sucrose and starch as organic substrates. Apart from hydrogen, variations in pH, volatile fatty acids, and solvent concentrations were also monitored. Initial pH was found to have a profound effect on both hydrogen production potential and hydrogen production rate. A mixed microbial culture was involved in the fermentation process with H-2, propionate, acetate, butyrate, and CO2 as major products. The lowest initial pH of 4.5 gave the highest specific hydrogen production potentials of 214.0 ml H-2/g chemical oxygen demand (COD) and 125.0 ml H-2/g COD for sucrose and starch respectively, but with the lowest specific hydrogen production rate. Although hydrogen production started earlier with the high production rate at a higher initial pH, the duration of the production was shorter. The rapid pH depletion could have caused a metabolic alteration of the microorganisms involved in hydrogen production, thereby resulting in the shift of intermediates production pathway [variation of the acetate/butyrate (HAc/HBu) ratio] and a consequent decrease in hydrogen production. The specific hydrogen production rate was highest for the pH range of 5.5-5.7. For the optimum pH range, the HAc/HBu ratio was in the range of 3-4 for both sucrose and starch. The findings of this study can be applied in the design of a high rate hydrogen bioreactor. (C) 2003 International Association for Hydrogen Energy. Published by Elsevier Ltd. All rights reserved.