The mannitol transport protein (EIImtl) carries out translocation with concomitant phosphorylation of mannitol from the periplasm to the cytoplasm, at the expense of phosphoenolpyruvate (PEP). The phosphoryl group which is needed for this group translocation is sequentially transferred from PEP via two phosphorylation sites, located exclusively on the C-terminal cytoplasmic domain, to mannitol. Oligonucleotide-directed mutagenesis was used to investigate the precise role of these sites in phosphoryl group transfer, by producing specific amino acid substitutions. The first phosphorylation site, His-554 (P1), was replaced by Ala, which renders the EII-H554A completely inactive in PEP-dependent mannitol phosphorylation, but not in mannitol/mannitol 1-phosphate exchange. The P2 site mutant, EII-C384S, was inactive both in the mannitol phosphorylation reaction and in the exchange reaction, due to replacement of the essential Cys-384 by Ser. Although EII-H554A and EII-C384S were both catalytically inactive in the PEP-dependent phosphorylation, EII-C384S was able to restore up to 55% of the wild-type mannitol phosphorylation activity with the EII-H554A mutant, indicating a direct phosphotransfer between two subunits. These phosphorylation data together with the data obtained from mannitol/mannitol phosphate exchange kinetics, after mixing EII-H554A and EII-C384S, indicated the formation of functionally stable heterodimers, which consist of an EII-H554A and an EII-C384S monomer.