The inhibition of cysteine biosynthesis in prokaryotes and protozoa has been proposed to be relevant for the development of antibiotics. Haemophilus influenzae O-acetylserine sulfhydrylase (OASS)， catalyzing l-cysteine formation， is inhibited by the insertion of the C-terminal pentapeptide (MNLNI) of serine acetyltransferase into the active site. Four-hundred MNXXI pentapeptides were generated in silico， docked into OASS active site using GOLD， and scored with HINT. The terminal P5 Ile accounts for about 50% of the binding energy. Glu or Asp at position P4 and， to a lesser extent， at position P3 also significantly contribute to the binding interaction. The predicted affinity of 14 selected pentapeptides correlated well with the experimentally determined dissociation constants. The X-ray structure of three high affinity pentapeptide-OASS complexes were compared with the docked poses. These results， combined with a GRID analysis of the active site， allowed us to define a pharmacophoric scaffold for the design of peptidomimetic inhibitors.