During translocation across the cytoplasmic membrane of Escherichia coli, glucose is phosphorylated by phospho-IIA^Glcand Enzyme IICB^Glc, the last two proteins in the phosphotransfer sequence of the phosphoenolpyruvate:glucose phosphotransferase system (PTS). Transient-state (rapid quench) methods were used to determine the second order rate constants that describe the phosphotransfer reactions (phospho-IIA^Glc to IICB^Glc to Glc) and also the second order rate constants for the transfer from phospho-IIA^Glc to molecularly cloned IIB(superGlc}, the soluble, cytoplasmic domain of IICB^Glc. The rate constants for the forward and reverse phosphotransfer reactions between IIA^Glc and IICB^Glc were 3.9x10⁶M^-1s^-1 and 0.31x10⁶M^-1s^-1 respectively, and for the physiologically irreversible reaction between [P]IICB^Glc and Glc was 3.2 x 10⁶M^-1s^-1. From the rate constants, the equilibrium constants for the transfer of the phospho-group from His 90 of [P]IIA^Glc to the phosphorylation site Cys of IIB^Glc or IICB^Glc were found to be 3.5 and 12 respectively. These equilibrium constants signify that the thiophospho-group in these proteins has a high phosphotransfer potential, similar to that of the phosphohistidinyl PTS proteins. In these studies, preparations of IICB^Glc were invariably found to contain endogenous, firmly bound Glc (estimated K^'_D = 10^-7M). The bound Glc was kinetically competent, and was rapidly phosphorylated, indicating that IICB^Glc has a random order, bi-bi, substituted enzyme mechanism. The equilibrium constant for the binding of Glc was deduced from differences in the statistical goodness of fit of the phosphotransfer data to the kinetic model.