In vitro kinetic measurements have been performed by using purified HPr, EI, and a membrane fraction of EII from the Escherichia coli phosphoenolypyruvate-dependent sugar transport system. These measurements reveal very large lag times in the formation of methyl alpha-glucoside phosphate which are a function of the EI and the EII concentrations. The lag times decrease with increasing concentrations of EI but they increase with increasing concentrations of EII. When EI, together with Mg2+ and phosphoenolpyruvate, is preincubated at 37 degrees C before starting the kinetic measurements, the lag time can be decreased or eliminated. We have shown that the process responsible for the lag time is the activation of EI by dimerization which is influenced by Mg2+ and phosphoenolpyruvate.