Chiral interactions are often important determinants for molecular recognition in chemistry and biochemistry. In order to determine whether the phospholipid backbone could be important for the conformational preference of membrane-spanning channels, we made use of the linear pentadecapeptide antibiotic gramicidin A (gA+) and a Trp-->Phe-substituted gA+ analogue, gramicidin M+ (gM+), as well as their enantiomers [gramicidin A- (gA-) and gramicidin M- (gM-), respectively]. All four analogues form conducting channels in planar bilayers formed from the dialkylphospholipids (R)- or (S)- dioleylphosphatidylcholine or from the diacylphospholipid (R)-dioleoylphosphatidylcholine. The characteristics of channels formed by the two gramicidin A enantiomers, or the two gramicidin M enantiomers, in membranes formed by either of the dioleylphosphatidylcholine enantiomers are indistinguishable. Similarly, channels formed by either pair of gramicidin enantiomers in dioleoylphosphatidylcholine bilayers are indistinguishable. We conclude that chiral interactions between gramicidin channels and the lipids in the host bilayer cannot be important determinants of gramicidin channel structure or function. The membrane/solution interface, therefore, seems to organize the channel structure because of the general characteristics of the nonpolar/polar transition at the interface rather than because of specific chemical interactions.