The mode of interaction of two pyrenylalanine residues which are introduced at positions 1 and 3 into gramicidine S, [Pya 1 , 3 ]GS, was investigated through absorption, CD, and steady-state and time-resolved fluorescence spectroscopy in organic and buffer solutions. CD spectra showed that the pyrene moieties could adopt an orientation to form a sandwich-type close pair on the two strands in a typical antiparallel β-structure in trifluoroethanol. Other spectroscopic studies demonstrated that the pyrenes of [Pya 1 , 3 ]GS in trifluoroethanol formed a specific intramolecular association of the pyrene pair, a sandwich-type dimer , in the ground state.