This study employed statistically based experimental designs to optimize fermentation conditions for hydrogen production from sweet sorghum syrup by anaerobic mixed cultures. Initial screening of important factors influencing hydrogen production, i.e., total sugar, initial pH, nutrient solution, iron (II) sulphate (FeSO4), peptone and sodium bicarbonate was conducted by the Plackett-Burman method. Results indicated that only FeSO4 had statistically significant (P <= 0.005) influences on specific hydrogen production (P-s) while total sugar and initial pH had an interdependent effect on P. Optimal conditions for the maximal P-s were 25 g/L total sugar, 4.75 initial pH and 1.45 g/L FeSO4 in which Ps of 6897 mL H-2/L was estimated. Estimated optimum conditions revealed only 0.04% difference from the actual P-s of 6864 mL H-2/L which suggested that the optimal conditions obtained can be practically applied to produce hydrogen from sweet sorghum syrup with the least error. (C) 2009 Professor T. Nejat Veziroglu. Published by Elsevier Ltd. All rights reserved.