Synthetic anion transporters (SATs) of the general type (n-C18H37)2N-COCH2OCH2CO-(Gly)3-Pro-(Gly)3-O-n-C7H15, 1, are amphiphilic peptides that form anion-conducting pores in bilayer membranes. To better understand membrane insertion, assembly and aggregation dynamics, and membrane penetration, four novel fluorescent structures were prepared for use in both aqueous buffer and phospholipid bilayers. The fluorescent residues pyrene, indole, dansyl, and NBD were incorporated into 1 to give 2, 3, 4, and 5, respectively. Assembly of peptide amphiphiles in buffer was confirmed by monitoring changes in the pyrene monomer/excimer peaks observed for 2. Solvent-dependent fluorescence changes that were observed for indole (3) and dansyl (4) side-chained SATs in bilayers showed that these residues experienced an environment between epsilon=9 (CH2Cl2) and epsilon=24 (EtOH) in polarity. Fluorescence resonance energy transfer (FRET) between 2 and 3 demonstrated aggregation of SAT monomers within the bilayer. This self-assembly led to pore formation, which was detected as Cl(-) release from the liposomes. The results of acrylamide quenching of fluorescent SATs supported membrane insertion. Studies with NBD-labeled SAT 5 showed that peptide partition into the bilayer is relatively slow. Dithionite quenching of NBD-SATs suggests that the amphiphilic peptides are primarily in the bilayer's outer leaflet. Images obtained by using a fluorescence microscope revealed membrane localization of a fluorescent SAT. Taken together, this study helps define the insertion, membrane localization, and aggregation behavior of this family of synthetic anion transporters in liposomal bilayers.