The antimicrobial peptides magainin 2 and PGLa isolated from the skin of the African clawed frog Xenopus laevis show marked functional synergism. We have proposed that the two peptides form a heterodimer composed of parallel helices with strong membrane permeabilizing activity [Hara, T., Mitani, Y., Tanaka, K., Uematsu, N., Takakura, A., Tachi, T., Kodama, H., Kondo, M., Mori, H., Otaka, A., Fujii, N., and Matsuzaki, K. (2001) Biochemistry 40, 12395-12399]. In this study, to elucidate the molecular mechanism of the synergy, we synthesized a chemically fixed heterodimer and investigated in detail the interaction of the hybrid peptide with bacteria, erythrocytes, and lipid bilayers. The hybrid peptide showed antimicrobial activity and membrane permeabilizing activity against negatively charged membranes, similar to or even stronger than those of a physical equimolar mixture of magainin and PGLa, indicating that the synergy is due to the formation of a parallel heterodimer. The heterodimer assumed a more oblique orientation than the component peptides. In contrast, the cross-linking of the two peptides significantly strengthened the action against erythrocytes and zwitterionic lipid bilayers by enhancing the affinity for membranes without changing the basic mode of action. Thus, the separate production of mutually recognizing peptides without cross-linking appears to be a good way to increase selective toxicity.