The sequence of peptides necessary to inhibit melittin-induced lysis was studied using 13 peptide analogues of the inhibitor Ac-IVIFDC-NH2. Although this inhibitor is a disulfide-linked dimer, inhibition was equally effective if the thiol SH was blocked or replaced by methionine or lysine. The substitution of phenylalanine with other aromatic residues preserved activity, as did the replacement of aspartic acid by asparagine. The results suggest that the cytolytic activity of melittin can be inhibited by a short peptide of four hydrophobic residues followed by two other nonspecific residues. Fluorescence studies showed that the inhibitor caused a blue shift in the Trp emission spectrum. A spin label attached to the N-terminus of the inhibitor significantly quenched the fluorescence. These data confirmed the involvement of Trp 19 with the inhibitor, also predicted by molecular modeling of the probable binding site. Density gradient studies with large unilamellar vesicles indicated that the inhibitor prevented melittin from reacting with the lipid bilayer.