Changes of bacterioplankton diversity in lake water were followed in triplicate, continuous‐flow experimental tanks. Most probable numbers (MPN) were obtained for 95 different carbon sources using BIOLOG plates and were used to characterize bacterioplankton diversity. During 70 days of incubation, MPN declined for 15 of the 95 substrates while three of 95 appeared to be newly used, indicating functional succession in the bacterioplankton. Total bacterial cell abundance was constant from day 7 to day 70 of the incubation period. The succession of species composition of phyto‐ and zooplankton was also observed and suggested some involvement by phyto‐ and zooplankton species in the changes of bacterioplankton diversity. Thus, BIOLOG‐based MPN assays is a simple but sensitive method for characterizing the changes in the bacterioplankton carbon utilization profile and is also useful for tracing the functional succession of bacterioplankton diversity within a community.