During a cycle of mannitol transport and phosphorylation, the phosphoryl group originating on P-enolpyruvate is transferred, consecutively, to two sites on the Escherichia coli mannitol-specific carrier (EIIMtl) before being placed on mannitol [Pas et al. (1988) Biochemistry (in press)]. The peptides constituting the two EIIMtl phosphorylation sites have been isolated and identified after labeling with [32P]-P-enolpyruvate. The first site is localized in peptide Leu 541-Lys 560. The hydrolysis characteristics of the phosphorylated peptide indicate that a histidine residue is phosphorylated. The second site is located in peptide Ile 380-Met 393, which contains the activity-linked cysteine (384) [Pas & Robillard (1988) Biochemistry (in press)]. The hydrolysis characteristics of the phosphopeptide indicate that Cys 384 is the site of phosphorylation.