GutR is a 95-kDa glucitol-dependent transcription activator that mediates the expression of the Bacillus subtilis glucitol operon. Glucitol allows GutR to bind tightly to its binding site located upstream of the gut promoter. In this study, a second functional role of glucitol is identified. Glucitol induces GutR to change its conformation and triggers GutR to bind ATP efficiently. After sequential binding of glucitol and ATP to GutR, GutR adopts a new conformation by forming a compact structure that is resistant to trypsin digestion. Under this condition, the ATP⅐glucitiol⅐GutR complex can dissociate slowly from the gutR-binding site (t1 ⁄2 ‫؍‬ 274 min). Interestingly, if ATP in the ATP⅐glucitiol⅐GutR complex is replaced by ADP, GutR adopts another conformation and can dissociate from the gutR-binding site even faster (t1 ⁄2 ‫؍‬ 82 min). In all these GutR-DNA binding studies in the presence of different ligands (glucitol, ATP, or ADP), only the off-rate is affected. The vital role of ATP in the GutRmediated transcription activation process is reflected by the poor transcription from the gut promoter with GutR(D285A) which has a mutation in the motif B of the putative ATP-binding site. A working model for this transcription activation process is presented.