Ladder-shaped polycyclic ethers (LSPs) exhibit potent toxicity due to their strong interactions to channel proteins. For example, brevetoxin binds sodium channels, and gambierol binds potassium channels. However the detail of this interaction has not been clarified yet. We estimated the hypothesis that LSPs interact with alpha helices of channel proteins through the hydrogen bond as figure 1. To prove this hypothesis, we attempt to prepare a double transmembrane peptide (A). This peptide was designed to have amphiphilic character, which may cause this peptide making a four alpha helix bundle like channel proteins. N-terminal peptide thioester and C-terminal cysteine peptide were synthesized by solid phase peptide synthesis. These peptides were connected by native chemical ligation subsequently to obtain peptide (A). Next, the optimal method of reconstitution in liposomes was investigated. As the result, the detergent removal method by dialysis using CHAPS was the best suited. In order to analyze the interaction between the peptide and lipid bilayer, gel filtration, digestion by a protease, and FRET experiments were conducted. For the positive and negative controls, peptide (B) and (C) were prepared by chemical synthesis. Peptide (B) is known as a transmembrane peptide, and peptide (C) is known to bind the surface of lipid bilayer. In the gel filtration, peptide (A) and LUV were eluted at the same time, indicating peptides to associate with liposomes. In the digestion by a protease, thermolysin, was used as a protease that cut the N side of hydrophobic residues. After treatment of peptide (A) reconstituted in liposome with thermolysin for 1 hour and 3 hours, the peptide MS peak was detected by LC-ESI MS. In contrast, the peptide not reconstituted in liposome was not detected after 1 hour digestion. However, any digested fragments of the peptide were not detected. A FRET experiment was planned, in addition. When FRET between fluorescent labeled phospholipids and the fluorescent group of peptide (F) is observed, it shows that the center of the peptide is located at the center of the lipid bilayer. As the result, the FRET induced fluorescence, though in a low intensity, was observed in the fluorescent labeled peptide (F) and peptide (B). FRET between the double transmembrane peptide and LSP (yessotoxin) will be similarly attempted to observe.