HPr of the bacterial phosphotransferase system is a histidine-containing phospho-carrier protein. It is phosphorylated at a single histidyl residue with phosphoenolpyruvate (PEP) and enzyme I and transfers the histidyl-bound phosphoryl group to a variety of factor III proteins. Recently, we described an HPr phosphorylated at a seryl residue (P-Ser-HPr), which is formed in an adenosine 5'-triphosphate dependent reaction catalyzed by a protein kinase [Deutscher, J., & Saier, M.-H., Jr. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 6790-6794]. Now we demonstrate that this P-Ser-HPr is an altered substrate of phosphorylated enzyme I and factor III proteins compared to unphosphorylated HPr. Thus, P-Ser-HPr of Streptococcus lactis is phosphorylated about 5000 times slower by PEP and enzyme I than HPr. The slow phosphorylation by PEP and enzyme I can be overcome when factor III protein specific for gluconate (factor III(Gct)) of Streptococcus faecalis is added. Most likely, a complex of P-Ser-HPr and factor III(Gct) is formed which then becomes phosphorylated as fast as free HPr. Factor III protein specific for lactose (factor III(Lac)) of Staphylococcus aureus also enhances the phosphorylation of P-Ser-HPr by enzyme I and PEP, but its effect is lower. Thus, P-Ser-HPr is phosphorylated 70-100-fold slower in the presence of factor III(Lac) than in the presence of factor III(Gct). The described interaction of P-Ser-HPr with enzyme I in the presence of different factor III proteins could account for the regulation of sugar uptake within the phosphotransferase system. Some of the phosphoenolpyruvate-dependent phosphotransferase system sugars like glucose are known to be taken up in preference to others, for example, lactose.