The phosphoenolpyruvate(P-pyruvate)-dependent sugar phosphotransferase system (PTS) is a transport and signal-transduction system which is almost ubiquitous in bacteria but does not occur in eucaryotes. It catalyzes the uptake and phosphorylation of carbohydrates and is involved in signal transduction, e.g. catabolite repression, chemotaxis, and allosteric regulation of metabolic enzymes and transporters. EI (Enzyme I of the PTS) is the first and central component of the divergent PTS (P-pyruvate -dependent sugar phosphotransferase system) phosphorylation cascade. Using immobilized combinatorial peptide libraries and phosphorimaging, heptapeptides and octapeptides were identified which selectively inhibit EI in vitro. The IC50 of the best peptides is 30 mu M which is close to the K-M (6 mu M) of EI for its natural substrate HPr (histidine containing phosphoryl carrier protein of the PTS). The affinity-selected peptides are better inhibitors than a peptide with the active-site sequence of HPr. The selected peptides contain several basic residues and one aromatic residue which do not occur in the active site bf HPr. The large proportion of basic residues most likely reflects charge complementarity to the strongly acidic active-site pocket of EI. Guanidino groups might facilitate by complexation of the phosphoryl group the slow phosphorylation of the peptide.