Key factors affecting biohydrogen production from waste glycerol and sludge by anaerobic mixed cultures were optimized using response surface methodology (RSM) with central composite design (CCD). Investigated parameters were waste glycerol concentration, sludge concentration, and the amount of Endo nutrient addition. Concentrations of waste glycerol and sludge had a significant individual effect on hydrogen production rate (HPR) (p <= 0.05). The interactive effect on HPR ( p <= 0.05) was found between waste glycerol concentration and sludge concentration. The optimal conditions for the maximum HPR were: waste glycerol concentration 22.19 g/L, sludge concentration 7.16 g-total solid (TS/L), and the amount of Endo nutrient addition 2.89 mL/L in which the maximum HPR of 1.37 mmol H-2/L h was achieved. Using the optimal conditions, HPR from a co-digestion of waste glycerol and sludge (1.37 mmol H-2/L h) was two times greater than the control (waste glycerol without addition of sludge) (0.76 mmol H-2/L h), indicating a significant enhancement of HPR by sludge. Major metabolites of the fermentation process were ethanol, 1,3-propanediol (1,3-PD), lactate, and formate. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.