This study evaluated the influences of inoculum-substrate ratio (ISR), volatile fatty acids (VFAs) and nicotinamide adenine dinucleotide, the reduced form (NADH) on hydrogen production during the anaerobic fermentation of Chlorella sp. in batch tests at 35 degrees C. The results indicated that the hydrogen concentration and lag time increased when ISR decreased, and the maximum hydrogen production and hydrogen content, 7.13 mL/g VS and 45.3%, respectively, were obtained when ISR was equal to 0.3. On the other hand, VFAs concentrations increased with the increase of hydrogen. The NADH increased while the daily output of hydrogen decreased as the fermentation carried on. The results suggested that ISR, VFAs, and NADH were important parameters for effective anaerobic hydrogen production using Chlorella sp. as substrate. (C) 2011 Elsevier Ltd. All rights reserved.