In our previous paper it was shown that the two C-terminal Gln residues of a C-terminal 15-residue fragment, Mel(12-26)(GLPALISWIKRKRQQ-NH2), of melittin and a series of individual substituted analogues might not involved in the interaction withbacterial membranes. In this paper, peptides with one and two Gln residues deletion, respectively, Mel(12-25) and Mel(12-24), weresynthesized and characterized. Both of the deletion peptides showed higher antimicrobial activities than the parent peptide, Mel(12-26). If both of the Gln residues of Mel(12-26) were respectively replaced by a hydrophilic amino acid Gly, the antimicrobial activity increasedslightly. If the Gln residue of Mel(12-25) was replaced by a hydrophobic amino acid Leu, the antimicrobial activity changed little, althoughthe substituted peptide possessed much higher hydrophobicity and higher α-helical conformation percentage in 1,1,1,3,3,3-hexafluoro-2-propanol/water determined by circular dichroism spectroscopy (CD) than the parent peptide. These results indicated that the two C-terminalresidues might be indeed not involved in the binding to bacterial membranes. The antimicrobial activity increasing with the residue deletionmay be caused by the decrease of the translational and rotational entropic cost of the binding of the peptides to bacterial membranes becauseof the lower molecular weights of the deletion peptides.