Waste sludge can be solubilized after S-TE (solubilization by thermophilic enzyme) pretreatment as the cryptic growth occurs at the expense of the cell lysate. The hydrogen production from S-TE sludge is greatly influenced by many factors. In this study, factors including pH, C/N, C/P, and Fe2+ affecting hydrogen production from S-TE sludge were optimized using uniform design. The optimum condition for maximum hydrogen yield of 68.4 ml H-2/g VSS (volatile suspended solid) could be predicted from regression model, and the optimum conditions were pH of 6.4, C/N ratio of 38, C/P ratio of 265, and Fe2+ concentration of 85 mg/L. There was interaction effect of factors on hydrogen production from S-TE sludge. Different pH, C/N, C/P and Fe2+ conditions could influence the VSS removal rate, carbohydrate and protein utilization. When the highest compositions of acetate and ethanol and lowest propionate were observed in metabolites, effective hydrogen production was also achieved. (C) 2013 Elsevier Ltd. All rights reserved.