In an attempt to identify the bioflocculation mechanisms, this study examines the role of calcium ions in flocculation of activated sludge. Two calcium specific chelants, ethylenebis (oxyethylenenitrilo)tetraacetic acid (EGTA) and sodium hexametaphosphate (HMT) are used to extract calcium ions. Both chemicals successfully extract the calcium ions from sludge structure, which is confirmed either by an increase in solution calcium concentration or by a decrease in calcium concentration in the sludge solid matrix. When calcium ions are removed from sludge, the physical properties of sludge change significantly. Filterability decreases, while both the turbidity of the supernatant and the solution carbohydrate concentration increase. The decrease in filterability and increase in turbidity indicate a flee breakup, and the increase in solution carbohydrate concentration gives evidence of the release of extracellular polymeric materials from the sludge flee structure. Application of both complexing agents (EGTA and HMP) creates similar changes in sludge properties. A sludge flee model is proposed which suggests that calcium ions help form colonies from individual microorganisms interacting with microbial extracellular polymers; following this they can further take part in bridging the microbial colonies together to form flocs. These interactions are believed to be among important biofloc formation mechanisms.