AbstractAerobic granulation represents an important bacterium‐to‐bacterium self‐immobilization process that has been exploited for the treatment of a wide spectrum of wastewaters, but the mechanism behind still remains unclear in a microbiological sense. This study investigated the possible involvement of ATP and autoinducer‐2 (AI‐2) in aerobic granulation. Results revealed that initiation of microbial aggregation is closely associated with the ATP content of biomass, whereas AI‐2 of biomass would be essential for maturation of aerobic granules. Furthermore, it was found that the AI‐2‐associated coordination of microorganisms in microbial aggregates would be biomass density dependent. This study clearly shows the involvement of ATP and autoinducer‐2 in aerobic granulation, and may be exploited further for enhancement or prevention of microbial aggregation in general, for example, rapid granulation for wastewater treatment or inhibition of biofouling in membrane bioreactor. Biotechnol. Bioeng. 2010;105: 51–58. © 2009 Wiley Periodicals, Inc.