Extracellular polymeric substances (EPS) play a key role in wastewater treatment, specifically flocculation, settling and treatment efficiency. Proteins are the predominant components in EPS and influence hydrophobicity and flocculation of sludge. In this paper, two-dimensional gel electrophoresis (2-DGE) technique was used to analyse protein profiles of EPS from a membrane biological reactor (MBR) fed either glucose or acetate. A highly reproducible 2-DGE protocol was developed achieving >95% of protein spots matches in repeated runs. Results also showed that proteins of EPS obtained from the activated sludge displayed a similar protein pattern between pH 4-7 with molecular weight 10-80 kD. Protein profiles of EPS extracted from activated sludge fed glucose and acetate showed similar distribution patterns for proteins, but further separation of proteins between pH 4-7 revealed distinctive differences between the specific proteins from these two carbon sources. In addition, information of 2-DGE protein profiles indicates little relationship between the physical and chemical analyses. It is apparent that 2-DGE is a much more powerful and a very useful tool in studying metabolisms of microbial communities at the level of metaproteomics.