BACKGROUND: Extracellular enzymes and chemical composition of sludge flocs affect the wastewater treatment capability. Here a fractionation protocol is presented describing the distributions of extracellular enzymes, proteins (PN) and polysaccharides (PS) in sludge floes. Sludge floc samples collected from 14 full-scale wastwater treatment plants (WWTPs), including those treating sewage, leachate and industrial wastewaters, were fractionated through centrifugation and ultrasound into five fractions: (1) supernatant; (2) slime; (3) loosely bound extracellular polymeric substances (LB-EPS); (4) tightly bound EPS (TB-EPS); and (5) pellets. RESULTS: The distributions of extracellular enzymes, PN and PS in different types of sludge floes were almost identical. Considerable quantities of alpha-amylase were bound with the pellet fraction, while the remainder was uniformly dispersed over the sludge matrix. Conversely, alkaline phosphatase, acid phosphatase and protease bound mainly with the pellet and TB-EPS fractions. CONCLUSION: The enzyme activities distributed in sludge floes achieved from 14 full-scale WWTPs revealed a non-wastewater-specific manner. This work for the first time demonstrated that the activities of enzymes correlated with the characteristics of the wastewaters treated, and raised the possibility of manipulating the hydrolysis reactions using process parameters. (C) 2008 Society of Chemical Industry