We have determined the overall stereochemical course of the reactions leading to the phosphorylation of D-mannitol by mannitol-specific enzyme II (EIIMtl) of the Escherichia coli phosphoenolpyruvate- (PEP) dependent phosphotransferase system (PTS). In the presence of enzyme I and HPr of the PTS, and of membranes containing EIIMtl, the phospho group from [(R)-16O,17O,18O]PEP was transferred to D-mannitol to form mannitol 1-phosphate with overall inversion of the configuration at phosphorus with respect to that of PEP. Since in the course of these reactions enzyme I and HPr are each covalently phosphorylated at a single site and inversion of the chiral phospho group from PEP indicates an odd number of transfer steps overall, transfer from phospho-HPr to mannitol via EIIMtl must also occur in an odd number of steps. Taken together with the fact that catalytically important phospho-EIIMtl intermediates have been demonstrated biochemically, our results imply that EIIMtl is sequentially phosphorylated at two different sites during phospho transfer from phospho-HPr to mannitol. This conclusion is consistent with the available evidence on phospho-EIIMtl intermediates and in particular with the recent report that two different phospho peptides can be isolated from the fully phosphorylated protein [Pas, H. H., & Robillard, G. T. (1988) Biochemistry 27, 5835-5839].