The performance of biological hydrolysis (BH) pretreatment on municipal secondary sludge was evaluated in this study. During 6-day BH at 42 degrees C (BH42), soluble chemical oxygen demand (sCOD) increased from 175.2 +/- 38.2 mg/L to 3314.5 +/- 683.4 mg/L; the dominant volatile fatty acid (VFA) was acetic acid, and its concentration increased from 41.5 +/- 2.1 mg/L to 786.0 +/- 133.2 mg/L. The extracted extracellular polymeric substances (EPS) from untreated secondary sludge contained three main fractions, and Fraction I gradually decreased from 133.9 kDa to 24.9 kDa during 6-day BH42. The BH pre-treatment at 42 degrees C and 55 degrees C both achieved more than 4-log reduction of total coliforms and 3-log reduction of E. coli. The BH pretreated secondary sludge at 15-day biochemical methane potential (BMP) test was comparable with the untreated secondary sludge after 30-day BMP, showing a significant enhancement on the acceleration of biogas production by BH pre-treatment.