The Escherichia coli BglF protein, also designated EIIbgl, is an enzyme II of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) that catalyzes transport and phosphorylation of beta-glucosides. In addition, BglF has the ability, unusual for an EII, to regulate the activity of a transcriptional regulator, BglG, by phosphorylating and dephosphorylating it according to beta-glucoside availability. Together, BglF and BglG constitute a novel sensory system. The membrane-bound sensor, BglF, has two phosphorylation sites: site 1 accepts a phosphoryl group from HPr and delivers it to site 2; site 2 delivers the phosphoryl group either to beta-glucosides or to BglG. Here, we provide several lines of evidence for the dimerization of BglF and for the occurrence of productive intersubunit phosphotransfer within the BglF dimers. (1) Two inactive BglF mutant proteins, one lacking phosphorylation site 1 and the other lacking site 2, complement one another to allow beta-glucoside utilization by bglF strains. (2) The pairs of mutant proteins complement one another in regulating BglG activity as a transcriptional antiterminator in vivo. (3) Only when they are present in the same membrane preparation do the mutant protein pairs efficiently transfer the phosphoryl group from HPr to beta-glucosides and to BglG in vitro. (4) Gentle extraction of cellular proteins followed by SDS-PAGE reveals the existence of BglF homodimers. A portion of the phosphorylated form of BglF can also be extracted from the membrane as a dimer. Dimerization is mediated by the membrane-bound IICbgl domain, as indicated by the dimerization of IICbgl by itself and of BglF derivatives that contain this domain. Since dimers persist in the presence of a reducing agent, they are apparently not held together by disulfide bonds. Rather, BglF dimerization might involve hydrophobic interactions between residues in the membrane-spanning domain. In addition, we show that BglF dimerization is not modulated by beta-glucosides and is therefore not part of the mechanism that diverts the phosphoryl group away from BglG to the transported sugar upon addition of beta-glucosides to the growth medium.