Membrane bioreactors have become a well-established alternative for conventional activated sludge systems with a filtration instead of a sedimentation step to separate the sludge from the purified water. Both separation processes, however, depend on good bioflocculation for their performance. Small activated sludge constituents are known to be detrimental in activated sludge filtration, while poor settling can often be attributed to improper floc formation. Therefore, the aim of this paper is to further unravel the relationship between bioflocculation and activated sludge separation processes. To this end, synthetic influents with different mono-over-polyvalent (M/P) cationic compositions are used to create activated sludges with different morphologies and bioflocculation states during a 178 days experiment. The activated sludges were characterized by microscopic image analysis, relative hydrophobicity, extracellular polymeric substances, and were subjected to sedimentation, dead-end and submerged crossflow filtration tests. The deflocculating effect of a high M/P ratio is affirmed, inducing the release of colloidal material that caused severe fouling. Sludge flocs became empty and did not settle well. In the low M/P reactor, overall a better separation performance was observed but a non-expected filamentous bulking episode caused bad settleability and bad crossflow filterability, while dead-end filterability was good since a porous, well structured fouling layer was formed. Hence, it is concluded that dead-end filtration tests cannot asses sludge filterability in MBRs and that big activated flocs cannot contribute to the cake layer and cannot ensure its porosity in this way. Hydrophobicity was generally higher in the low M/P reactor but this might be attributed to an abundance of filaments during part of the testing period. No conclusive information could be extracted regarding the role of EPS, thus the use of EPS as a fouling or bioflocculation indicator is (again) questioned. (C) 2014 Elsevier B.V. All rights reserved.