The effects of extracellular polymer (ECP) production and electrostatic properties of the substrates on the process of granulation were studied in up-flow anaerobic sludge blanket (UASB) reactors using three kinds of substrates, glucose, skim milk and mixed volatile fatty acids (VFAs). The study confirmed the important contribution of ECP-bonding to granulation and proved the effectiveness of slight overloading in stimulating ECP production, which shortened the period of granulation. Investigations on the surface charge of the substrates and sludge revealed that the different substrates had different surface properties, which affected the granulation process directly. Based on the DLVO theory that the repulsion forces between particles can be whittled down by adding oppositely charged ions, ECP content and the surface charges of substrates were believed to be the important triggering forces for the anaerobic granulation in the UASB reactors. In the process of bacterial adhesion, the substrates functioned as external electrolytes and the ECP materials worked as the high molecular flocculants. Both surface properties and ECP content were closely related to the substrates and the running conditions. As a result, the required granulation time and the behaviour of granules were dependent on the running conditions and the substrates themselves. (C) 2005 Elsevier Ltd. All rights reserved.