Effluent organic matter (EfOM) has been considered by many to play an important role in fouling of RO membranes used for wastewater reclamation. However, due to their heterogeneous composition, which is a mixture of structurally complex aquatic humic substances (AHS), soluble microbial products (SMP) or extracellular polymeric substances (EPS) and other poorly defined organic compounds, the fractional component(s) or physical-chemical properties responsible for the fouling phenomenon are still not well understood. This study aims to obtain a better understanding of interactions between fractional components of EfOM and RO membranes and attempts to identify the most influential fraction(s) or physical-chemical properties governing the fouling process. Four EfOM fractions were isolated and fractionated from OF prefiltrated treated effluent based on hydrophobicity and charge characteristics. EPS was extracted from the biological treatment stage to assess their fouling potential on RO membranes via well-controlled laboratory-scale experiments. The individual organic fractions were rigorously characterized in terms of physico-chemical properties. A clear correlation was observed between the physico-chemical properties of EfOM fractions and their fouling potential. Under hydrodynamic and chemical conditions typical of commercial applications, the hydrophilic neutral fraction, mainly composed of small size carbohydrates, resulted in the highest flux decline and exhibited highest affinity towards the membrane. EPS biopolymers, to which great importance has been associated with regard to causing RO organic fouling, resulted in less fouling than hydrophilic carbohydrates. Although EPS biopolymers tended to accumulate on the membrane in much higher quantities, the cake layer formed was found to constitute a much lower resistance towards filtration and has a much lower membrane affinity, probably due to their large molecular sizes. The contribution of AHS and other hydrophilic fractions to membrane fouling was found to be much lower as compared to hydrophilic carbohydrates and EPS biopolymers. (C) 2009 Elsevier B.V. All rights reserved.