The factors that change the microbial distribution and consequently the flotation of brewery granules were investigated using laboratory-scale upflow anaerobic sludge blanket (UASB) reactors and the fluorescence in situ hybridization (FISH) method. The startup operations of laboratory-scale UASB reactors fed with acetate-based synthetic wastewater, in which the loading rate was maintained at 0.1 gCOD/gVSS/d (Run 1) and increased in a stepwise manner from 0.1 gCOD/gVSS/d to 1.0 gCOD/gVSS/d (Run 2), generated methanogen colonies near the granule surface, while the overloading operation at 1.0 gCOD/gVSS/d from the startup (Run 3) resulted in the formation of methanogen colonies deep in the granules. In each run, a proportion of the granules floated when overloaded at 2.0 gCOD/gVSS/d and circulation was stopped. The ratio of floating granules increased as the methanogen-growing region increased. On the other hand, the Bacteria layer on the granule surface, which is also considered as a possible cause of granule flotation, was not formed by the inflow of other organic acids such as propionate and lactate. Glucose caused formation of a 5-mum-thick surface Bacteria layer, but the granules were still resistant to flotation. Interfusing of air under glucose feeding caused the formation of a Bacteria layer over 50 mum thick leading to granule flotation.