Thermophilic hydrolysis of grass silage (GS) at 55 C with organic loading rates (OLRs) of 6.5, 5, 2.5 and 1.0 kg VS m3 days1 and hydraulic retention times (HRT) of 10, 6, 4 and 2 days were evaluated in 12 glass bioreactors side by side. The hydrolytic process was measured by variation in pH, volatile solids (VS), VS destruction, soluble chemical oxygen demand (sCOD), hydrolysis and acidification yields. Biological methane potential (BMP) assays were carried out to measure the upper limit for methane production of grass silage with different hydrolytic pretreatments at mesophilic temperature (37 C). The optimum methane yield of 368 LN CH4 kg1 VS was obtained at an OLR of 1 kg VS m3 days1 and a HRT of 4 days, showing an increase of 30% in the methane potential in comparison to non-hydrolysed GS.