Combined pretreatment of heat treatment with alkaline condition was applied to the sewage sludge in order to acquire the effective method for sludge solubilization and bio-hydrogen production. Solubilization ratio of the sludge with heat treatment at alkaline condition (pH 13) was as high as 85.0%, resulting in an increase of COD concentration in aqueous solution, but the ratio of sludge with heat treatment or alkaline treatment alone was 32.2% and 56.3%, respectively. During the fermentation of the pretreated sludge, a significant increase of hydrogen production was observed with a low strength of ammonia, showing that ammonia in the aqueous phase could inhibit bio-hydrogen production. Klebsiella, Enterobacter or Clostridium genus were mostly related on the hydrogen production. (C) 2012 Elsevier Ltd. All rights reserved.