AbstractIn order to resolve whether gramicidin A channels are formed by right‐ or left‐handed β‐helices, we synthesized an optically reversed (or mirror image) analogue of gramicidin A, called gramicidin A−, to test whether it forms channels that have the same handedness as channels formed by gramicidin M−(F. Heitz et al., Biophys. J. 40:87–89, 1982). In gramicidin M−the four tryptophan residues have been replaced with phenylalanine, and the circular dichroism (CD) spectrum therfore reflects almost exclusively contributions from the polypeptide backbone. The CD spectrum of gramicidin M−in dimyristoylphosphatidylcholine vesicles is consistent with a left‐handed helical backbone folding motif (F. Heitz et al., Biophys. Chem. 24:149–160, 1986), and the CD spectra of gramicidins A and A−are essentially mirror images of each other. Based on hybrid channel experiments, gramicidin A−and M−channels are structurally equivalent, while gramicidin A and A−channels are nonequivalent, being of opposite helix sense. Gramicidin A−channels are therefore left‐handed, and natural gramicidin A channels in phospholipid bilayers are