Elucidating the function of the microbial community in activated sludge is the key to understanding its treatment mechanism. Although the removal of organic matter is one of the major functions of a microbial community, the substrate utilization of each microorganism remains to be fully elucidated. In this study, DNA stable isotope probing (DNA-SIP) was applied to the analysis of activated sludge collected from a full-scale wastewater treatment plant to identify glucose- and acetate-assimilating bacteria. Activated sludge was fed with ¹³C-labelled glucose and ¹³C-labelled acetate individually and incubated for 60 hours. Two different substrate concentrations (100 mgC·L^-1, 1000 mgC·L^-1) with the same loading (300 mgC) were tested for glucose, while only one substrate concentration (100 mgC·L^-1, 300 mgC of loading) was tested for acetate. The DNA-SIP experiment clearly revealed that glucose was assimilated by bacteria closely related to Aeromonas, Enterobacteriaceae, and Tolumonas regardless of substrate concentration or activated sludge collected on a different day. On the other hand, a different group of bacteria closely related to Acidovorax, Acinetobacter, and Dechloromonas was identified as acetate-assimilating bacteria. Although specific glucose- and acetate-assimilating bacteria responded to glucose and acetate, most of them were not predominant in the original sludge.