The photophysics of linear Aib-based hexapeptides (Aib = alpha-aminoisobutyric acid) of general formula Ac-Toac-(Aib)(n)-Trp-(Aib)(r)-OtBu (TnTrp), where n + r = 4, and Toac and Trp are a nitroxide spin labeled C-alpha,C-alpha-disubstituted glycine and tryptophan, respectively, were investigated in methanol and dioxane solutions by steady-state and time-resolved fluorescence measurements. Another hexapeptide, i.e., Boc-(S)Bin-Ala-Aib-Toac-(Ala)(2)-OtBu (T-Bin), carrying the same nitroxide derivative and a binaphthyl as the fluorophore was also studied by the same techniques. Quenching of the excited tryptophan or binaphthyl chromophore proceeds on a time scale from subnanoseconds to a few nanoseconds, depending on the conformers distribution in solution. CD and IR spectral patterns in methanol or CDCl3 suggest that the backbone of the peptides examined is in the 3(10)-helical conformation, thus preserving the structural features of the crystal state, as earlier determined by X-ray diffraction measurements. The fluorescence results were satisfactorily described by a dipole-dipole interaction mechanism, in which electronic energy transfer takes place from the excited tryptophan or binaphthyl to Toac, provided the mutual orientation between the fluorophore and Toac is taken into account. This implies that interconversion among conformational substates is slow on the time scale of the transfer process. Molecular mechanics calculations coupled with time decay data allowed us to build up the most probable structures of these peptides in methanol solution.