This study examines enzyme hydrolysis, a mild, effective, but a rarely used method of extracellular polymer extraction, in removing polymers from mixed culture activated sludge flocs. Two carbohydrate specific enzymes (a-amylase and cellulase) and a protein specific enzyme (proteinase) are used during the study. First, the kinetic aspect is investigated, then enzyme dose optimization is carried out on laboratory grown activated sludge samples cultured at solids retention times (SRT) of 4 and 20 days. A more commonly used cation exchange resin (CER) extraction technique is also employed for comparison purposes. Results indicate that the extraction of extracellular polymers by enzymes is a rather quick process reaching equilibrium within only a few hours. As the doses of enzymes are increased, the extracted polymer quantities increase up to a certain dose, beyond which not much extraction is observed. The method does not cause any significant cell lysis as measured by the viable cell counts. Carbohydrate-hydrolyzing enzymes extract small amount of proteins along with the carbohydrates and protein-hydrolyzing enzyme extracts some carbohydrates together with the proteins, indicating that proteins and carbohydrates exist bound to each other in the extracellular polymer network of sludge. Enzyme extraction generally gives a lower estimate of polymers compared to the CER method, but correctly detects the trends in the polymer quantity. (c) 2006 Elsevier Ltd. All rights reserved.