The Escherichia coli BglF protein catalyzes transport and phosphorylation of β-glucosides. In addition, BglF is a membrane sensor which reversibly phosphorylates the transcriptional regulator BglG, depending on β-glucoside availability. Therefore, BglF has three enzymatic activities: β-glucoside phosphotransferase, BglG phosphorylase, and phospho-BglG (BglG- P) dephosphorylase. Cys-24 of BglF is the active site which delivers the phosphoryl group either to the sugar or to BglG. To characterize the dephosphorylase activity, we asked whether BglG-P can give the phosphoryl group back to Cys-24 of BglF. Here we provide evidence which is consistent with the interpretation that Cys-24-P is an intermediate in the BglG-P dephosphorylation reaction. Hence, the dephosphorylation reaction catalyzed by BglF proceeds via reversal of the phosphorylation reaction.