The optimal condition for hydrogen production using Clostridium beijerinckii PS-3 was at an initial pH of 6.3–6.7 and cultivation at room temperature (30±2ºC). Among various types of substrate tested, the hydrogen yield of 1.73 mol/mol-xylose was about 1.2, 1.6 and 5.6 fold higher than those of glucose, starch and cellulose, respectively. From the xylose concentrations (5-30 g/L) tested, the optimum value was 10 g/L, giving the cumulative hydrogen production and hydrogen yield of 3254 ml/L and 2.18 mol/mol-xylose, respectively. Soluble metabolites from xylose were composed mostly of butyric acid (39.0-48.7mM), acetic acid (35.0-39.9 mM), ethanol (30.2-35.3 mM) and propionic acid (4.9-6.5 mM).