The aggregation of macrocyclic oligocholates with introverted hydrophilic groups and aromatic side chains was studied by fluorescence spectroscopy and liposome leakage assays. Comparison between the solution and the membrane phase afforded insight into the solvophobically driven aggregation. The macrocycles stacked over one another in lipid membranes to form transmembrane nanopores, driven by a strong tendency of the water molecules in the interior of the amphiphilic macrocycles to aggregate in a nonpolar environment. The aromatic side chains provided spectroscopic signatures for stacking, as well as additional driving force for the aggregation. Smaller, more rigid macrocycles stacked better than larger, more flexible ones because the cholate building blocks in the latter could rotate outward and diminish the conformation needed for the water-templated hydrophobic stacking. The acceptor–acceptor interactions among naphthalenediimide (NDI) groups were more effective than the pyrene–NDI donor–acceptor interactions in promoting the transmembrane pore formation of the oligocholate macrocycles.