Relationships of bacterial populations and extracellular polymer substances (EPS) to dewaterability of activated sludge were studied on three laboratory-scale activated sludge reactors fed with synthetic wastewater. Dewaterability of activated sludge was evaluated by a novel method developed by the authors, in which small amount of sludge was centrifugally dewatered, and its water content was measured. Bacterial populations during the reactor operation were analyzed by the polymerase chain reaction/terminal-restriction fragment length polymorphism (PCR/T-RFLP) targeted at a partial 16S rRNA gene. Extracellular polymeric substances (EPS) were extracted using cation exchange resin (CER), and polysaccharides and total protein in EPS were determined. Some of the dominant terminal-restriction fragments (T-RFs) were observed to have significant relationships with dewaterability of sludge, and it was suggested that bacterial species corresponding to those peaks significantly affected dewaterability. On the other hand, significant relationships were not found between EPS concentration and dewaterability of sludge.