Six amphiphilic heptapeptides with the structure (C18H37)2NCOCH2OCH2CO-(Gly)3-Pro-(Gly)n-(Glx)-(Gly)m-O(CH2)6CH3, in which Glx represents glutamic acid or its benzyl ester and n+m=2, have been studied. In addition, the glutamate residue in the GGGPGGE sequence was esterified by fluorescent 1-pyrenemethanol. These compounds insert into phospholipid bilayers and form anion-conducting pores. Hill plots based on carboxyfluorescein release indicate that the pores are at least dimeric. Studies that involved ion-selective electrode techniques showed that transport of chloride varied with the position of glutamate within the peptide chain and whether glutamic acid was present as the free acid or its benzyl ester. Chloride transport activity was significantly higher for the glutamate esters than for free carboxylates irrespective of the glutamate position. Activity was highest when the glutamate residue in approximately (Gly)3-Pro-(Xxx)3 approximately was closest to the C terminus of the peptide. A fluorescent pyrene residue was introduced to probe the aggregation state of the amphiphile. The selectivity of the pore for Cl(-) over K+ was maintained even when the carboxylate anion was present within it. Complexation of Cl(-) by the ionophoric peptides was confirmed by negative ion mass spectrometry. Planar bilayer voltage clamp experiments confirmed that pores with more than one conductance state may form in these dynamic, self-assembled pores.