Mastoparan B (MP-B), an amphiphilic alpha-helical peptide newly isolated from the hornet Vespa basalis, was studied in comparison with mastoparan (MP), in terms of interaction with the phospholipid bilayer and of hemolytic and antimicrobial activity. The amphiphilic structure of MP-B has more hydrophilic amino acid residues in the hydrophilic surface than that of MP. Although each peptide had a considerably different effect on the interaction with lipid bilayers (e.g., their conformation in the presence of acidic and of neutral lipids and dye-release ability from the encapsulated liposomes), on the whole the interaction mode was similar. MP-B caused a change in the shape of erythrocytes from normal discoid to a crenated form (named echinocytes). MP exhibited strong activity against gram-positive bacteria but not against gram-negative ones. Contrary to this, MP-B showed both strong activity against gram-positive bacteria and potent activity against gram-negative bacteria. Whereas both peptides have almost the same residues on the hydrophobic side, the difference in the hydrophilic surface area on the molecules seems to lead to the subtle change in its interaction with membranes, resulting in the alteration of biological activity.