Bacterial biomass was characterised in supernatants from activated sludge from a nutrient removal plant after settling before and after applying gentle shear (G approximately 600 s(-1)). Free-swimming and floc-associated bacteria were quantified by microscopy and their identity was determined by fluorescence in-situ hybridization (FISH). Total cell numbers in the supernatant after settling ranged within 2-9 x 10(7)cells/mL. Most cells (60-70%) were associated with microcolonies or small flocs, which made up 5-10% of the total number of particles. The remaining 30-40% of the cells corresponded to free-swimming, single cells. The small flocs in the supernatants (diameter=2.5-35 microm) accounted only for approximately 1% of the total number of particles; however, they greatly contributed to the total volume of biomass in suspension (57% and 75%). The shear applied (G approximately 600 s(-1)) induced some floc detachment and higher cell numbers in the supernatants (10-70 x 10(7)cells/mL). The identity of bacteria in suspension was as diverse as that in the settled sludge; however, bacteria belonging to Planctomycetes, Firmicutes and Deltaproteobacteria were in higher abundance in the sludge supernatants and were enriched in the supernatants due to gentle shear. Potentially active bacteria were quantified based on the ratio of the number of cells fluorescing with the EUBmix gene probe targeting most bacteria to the total number of cells stained with DAPI. Lower ratios of EUBmix to total cells were measured in the supernatants (50%) than in the settled sludge (80%), suggesting that cells in the dispersed fraction of the sludge were potentially less active than those in the average settleable floc. In conclusion, the attachment properties of bacteria in activated sludge were different among groups, rendering floc fractions more susceptible to detachment and suspension depending on their abundance and activity level.