Abstract Activated sludge flocs are known to deflocculate under short-term anaerobic conditions, but little is known about possible reflocculation under subsequent aerobic conditions. When activated sludge flocs from two wastewater treatment plants deflocculated under anaerobic conditions with well-defined shear conditions, they could be almost, but not completely, reflocculated by aeration for 1–2 h under the same shear conditions. If the biological activity was reduced by adding azide, chloramphenicol or by decreasing the temperature, no or only very little reflocculation took place. This indicated that the reflocculation was under direct or indirect microbial control. Only a small part of the reflocculation was due to improved flocculation properties obtained by oxidation of Fe(II) to Fe(III), which is a better flocculant. Fe(II) was produced under the anaerobic conditions by microbial iron reduction, and it was oxidized to Fe(III) within less than one hour after the aeration was started. However, by comparing two different sludges with different capabilities for iron reduction, iron oxidation and responses to substrate addition, it was found that the aerobic biological activity most likely was of greatest significance for the observed reflocculation and floc formation under aerobic conditions. This was further supported by adding organic substrates (glucose or ethanol) during the aerobic reflocculation phase, which promoted reflocculation. However, some substrates had the opposite effect (acetate and lactate), where a deterioration of the reflocculation was observed, probably due to different responses from different groups of microorganisms in the sludges.