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Brevinin 1E, consisting of 24 amino acid residues, from Rana esculenta has potent antimicrobial and hemolytic activity. From a structural point of view, this peptide has a N-terminal hydrophobic region, a proline hinge region in the middle and a C-terminal loop region delineated by an intra-disulfide bridge, which is a common structural feature of antimicrobial peptides from Rana species. To investigate the structural features for antimicrobial and hemolytic activity, truncated and linearized brevinin 1E amides were synthesized and characterized. A deletion of three amino acids from the N-terminal region did not greatly affect antimicrobial activity but dramatically reduced hemolytic activity. The contribution of the intra-disulfide bridge to antimicrobial and hemolytic activity was somewhat different between brevinin 1E amide and truncated fragments. In brevinin 1E amide, the elimination of the intra-disulfide bridge did not greatly affect antimicrobial and hemolytic activity whereas the elimination of the intra-disulfide bridge in the truncated fragments did not decrease antimicrobial activity but did decrease hemolytic activity. Circular dichroism spectra and the retention time on the C18 reverse phase column revealed that the intra-disulfide bridge (i, i+6) formed an amphipathic loop which increased hydrophobicity and helped to induce the alpha-helical structure in the membrane-mimetic environment. Even though the intra-disulfide bridge and the N-terminal region were responsible for the alpha-helical structure and hydrophobicity, these two structural features were not essential for antimicrobial activity. The hemolytic activity of brevinin 1E amide and its analogs also correlated well with the retention time rather than the alpha-helicity.