In order to investigate why OSA (oxic-settling-anaerobic) process produces less sludge than CAS (conventional activated sludge) process, sequential cultivation through 1 st aerobic-anaerobic-2 nd aerobic conditions, were carried out. Then, the intracellular concentrations of adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD and NADH), and nicotinamide adenine dinucleotide phosphate (NADP and NADPH) were monitored for these three stages. Results showed that the concentrations of these energy substances rapidly decreased through time in both aerobic and anaerobic conditions but the anaerobic culture contained the lower energy level than aerobic culture. The 2 nd aerobic culture that experienced anaerobic condition showed lower concentration of these energy substances than those of the 1 st aerobic culture. Meanwhile, the anaerobic culture corresponding to the sludge holding stage of OSA was subjected to different soluble chemical oxygen demand (SCOD) levels, detention time, and temperature to evaluate the effects of these variations on the energy level difference between the 1 st and 2 nd aerobic stages. The lower the SCOD concentration, the longer detention time; and the higher temperature in the anaerobic stage tended to further reduce the intracellular level of the 2 nd aerobic culture. On the average, the intracellular energy level of the anaerobic and 2 nd aerobic stage were 57.73% and 39.12% of the 1 st aerobic culture, respectively. These indicated that the insertion of an anaerobic stage between two aerobic stages could lower the intracellular energy levels, hence the lower the sludge in OSA than CAS process. Moreover, manipulation of the operating conditions of the intervening anaerobic stage can change intracellular energy levels thereby controlling sludge production.