A macrocyclic and a linear trimer of a facially amphiphilic cholate building block were labeled with a fluorescent dansyl group. The environmentally sensitive fluorophore enabled the aggregation of the two oligocholates in lipid membranes to be studied by fluorescence spectroscopy. Concentration-dependent emission wavelength and intensity revealed a higher concentration of water for the cyclic compound. Both compounds were shown by the red-edge excitation shift (REES) to be located near the membrane/water interface at low concentrations, but the cyclic trimer was better able to migrate into the hydrophobic core of the membrane than the linear trimer. Fluorescent quenching by a water-soluble (NaI) and a lipid-soluble (TEMPO) quencher indicated that the cyclic trimer penetrated into the hydrophobic region of the membrane more readily than the linear trimer, which preferred to stay close to the membrane surface. The fluorescent data corroborated with the previous leakage assays that suggested the stacking of the macrocyclic cholate trimer into transmembrane nanopores, driven by the strong associative interactions of water molecules inside the macrocycles in a nonpolar environment.