Tryptophans at positions 4 and 7 of compstatin, a peptide complement inhibitor, are crucial for its interaction with C3. However, the nature of their involvement has not been studied to date. Here we investigate the molecular forces involved in the C3-compstatin interactions, mediated by aromatic residues, by incorporating in these two positions various tryptophan analogues (5-methyltryptophan, 5-fluorotryptophan, 1-methyltryptophan, and 2-naphthylalanine) and assessing the resulting peptides for activity by enzyme-linked immunosorbent assay (ELISA) and binding by isothermal titration calorimetry (ITC). Of all the compstatin analogues, peptides containing 1-methyltryptophan at position 4 exhibited the highest binding affinity (Kd = 15 nM) and activity (IC50 = 0.205 microM), followed by a peptide containing 5-fluorotryptophan at position 7. Our observations suggest that hydrophobic interactions involving residues at position 4 and the hydrogen bond initiated by the indole nitrogen are primarily responsible and crucial for the increase in activity. These findings have important implications for the design of clinically useful complement inhibitors.