Bio-hydrogen production from sweet sorghum stalk using mixed anaerobic sludge was reported in this paper. Batch tests were carried out to analyze influences of several environmental factors on yield of H-2 from sweet sorghum stalk under constant mesophillic temperature (36+/-1 degrees C). The experimental results showed that, for the raw stalk, the cumulative hydrogen yield was 52.1 ml g(-1). TVS with utilization percentages of sugars, hemi-cellulose and cellulose in the stalk being 89.12, 15.23 and 13.89% respectively; whereas for the stalk pretreated by 0.4% NaOH solution at room temperature for 24h, the cumulative hydrogen yield was 127.26 ml g(-1). TVS with utilization percentages of sugars, hemi-cellulose and cellulose being 99.17, 53.64, 41.56%, respectively. The hydrogen content in the biogas was about 53% while the methane content was less than 4% throughout the study. Besides hydrogen and methane, the main metabolic products detected were ethanol, propionate and butyrate. The experimental results suggested that the alkalization pretreatment of the substrate plays a crucial role in the conversion of the sweet sorghum stalk wastes into bio-hydrogen by the mixed anaerobic sludge. Copyright (C) 2009 John Wiley & Sons, Ltd.