In natural environments, bacteria fluctuate between growth as motile cells and growth as sessile, biofilm-forming cells. However, what controls the transition between these two-growth modes in Bacillus subtilis is not well understood yet. The degU mutation prevents both flagellum formation and biofilm formation, suggesting that one of the transition mechanisms may underlie regulation of the DegU activity. The expression profiles of DegU-regulated genes differed; flagellar genes and several unknown genes were expressed during the exponential phase, whereas other genes were induced in the stationary phase. The degS mutation did not affect transcription of the flgB-sigD operon, but reduced transcription of sigma(D)-dependent flagellar genes, degU and other DegU-regulated genes. In addition, the degQ mutation did not affect transcription of flagellar genes but reduce transcription of other DegU-regulated genes. Purified DegQ protein stimulated phosphotransfer from phospho-DegS to DegU in vitro. Moreover, DegU binds the promoter region of flgB with a high affinity, whereas DegU binds to the promoter regions of other DegU-regulated genes with a low affinity and in a DegS-dependent manner. Taken together, we propose that a gradual increase in DegU and phospho-DegU levels induces a transition from growth as motile cells to growth as sessile, biofilm-forming cells.