Polyenemacrolides such as amphotericin B (AmB) were thought to assemble together and form an ion channel across plasma membranes. Their antimicrobial activity has been accounted for by this assemblage, whose stability and activity are dependent on sterol constituents of lipid bilayer membranes. The structure of this channel-like assemblage formed in biomembranes has been a target of extensive investigations for long time. For the first step to this goal, we prepared several AmB dimers with various linkers and tested for their channel-forming activity. Among these, AmB dimers that bore an aminoalkyl-dicarboxylate tether covalently linked between amino groups of AmB showed potent hemolytic activity. Furthermore, K+ influx actions monitored by pH of liposome lumen by 31P NMR revealed that the dimers formed the molecular assemblage similar to that of AmB in phospholipid membrane. Judging from changes in 31P NMR spectra, the dimers appeared to induce "all-or-none" -type ion flux across liposome membrane in the presence of ergosterol, which suggested that ion-channel formed by ergosterol/dimer is similar to that of AmB. With these data in hand, we are now trying to elucidate the structure of the ion-channel complex by making the labeled conjugates of AmB for NMR measurements.