We have previously reported that glucose can be phosphorylated by phospho-HPr and two sugar-specific pairs of proteins of the Escherichia coli and Salmonella typhimurium phosphoenolpyruvate:glycose phosphotransferase system. Each of the sugar-specific complexes comprises two proteins, lipid, and divalent cation, and each is present in membranes isolated from wild type cells. For reasons described in this report, one of the complexes is designated IIGlc and the other IIMan. The IIMan complex has previously been separated into its protein components, II-A and II-B (Kundig, W., and Roseman, S. (1971) J. Biol. Chem. 246, 1407-1418), while the accompanying reports describe dissociation of the IIGlc complex into its components, IIIGlc and II-BGlc. Curtis and Epstein (Curtis, S. J., and Epstein, W. (1975) J. Bacteriol. 122, 1189-1199) first showed that there are two phosphotransferase systems in whole cells responsible for glucose uptake and obtained the respective mutants, now designated ptsG and ptsM. The present studies provide kinetic conditions for assaying each activity separately (in vivo and in vitro), when both are present in the same membrane preparation. The IIGlc system is responsible for the uptake and phosphorylation of glucose and methyl alpha-glucoside, whereas the IIMan system is less specific and utilizes glucose, mannose, and 2-deoxyglucose. With high sugar concentrations in vitro, IIMan is also capable of phosphorylating methyl alpha-glucoside, fructose, and N-acetylmannosamine, while IIGlc phosphorylates fructose and mannose. The in vivo transport results were qualitatively consistent with the in vitro phosphorylation results, and several of the kinetic parameters also showed good quantitative agreement. The levels of the two activities depended on the growth conditions. In addition, transport studies showed that initial uptake rates of methyl alpha-glucoside and steady state levels of this analogue depended on the energy state of the cells and that these two parameters did not necessarily change in the same direction when metabolic inhibitors were used. A series of E. coli and S. typhimurium mutants were characterized both with respect to their ability to transport the glucose analogues and to phosphorylate them in vitro. The original mutants of Curtis and Epstein, ptsG and ptsM, were found to be defective in II-BGlc and the IIMan complex, respectively.