In the present study, mesophilic CH4 production from grass silage in a one-stage process was compared with the combined thermophilic H-2 and mesophilic CH4 production in a two-stage process. In addition, solid and liquid fractions separated from NaOH pre-treated grass silage were also used as substrates. Results showed that higher CH4 yield was obtained from grass silage in a two-stage process (467 ml g(-1) volatile solids (VS)original) compared with a one-stage process (431 ml g(-1) VSoriginal). Similarly, CH4 yield from solid fraction increased from 252 to 413 ml g(-1) VSoriginal whereas CH4 yield from liquid fraction decreased from 82 to 60 ml g(-1) VSoriginal in a two-stage compared to a one-stage process. NaOH pre-treatment increased combined H-2 yield by 15% (from 5.54 to 6.46 ml g(-1) VSoriginal). In contrast, NaOH pre-treatment decreased the combined CH4 yield by 23%. Compared to the energy value of CH4 yield obtained, the energy value of H-2 yield remained low. According to this study, highest CH4 yield (495 ml g(-1) VSoriginal) could be obtained, if grass silage was first pre-treated with NaOH, and the separated solid fraction was digested in a two-stage (thermophilic H-2 and mesophilic CH4) process while the liquid fraction could be treated directly in a one-stage CH4 process. (C) 2009 Elsevier Ltd. All rights reserved.