The structure, spectroscopy, and photophysical behavior of a series of hairpin-forming conjugates possessing a 5'-tethered N-alkylpyrenecarboxamide chromophore have been investigated. Comparison of the NMR spectra of the conjugates and analogs lacking the tethered pyrene indicates that the pyrene does not behave as ail end-capping group but rather is intercalated between the two terminal hairpin base pairs. An intercalated structure is also consistent with the thermodynamic parameters for hairpin formation and the steady state and transient spectral properties of the conjugates. Quenching of the pyrene fluorescence and transient absorption spectra is observed only when guanine is located in one of the two terminal base pairs and is attributed to hole injection from singlet pyrene to guanine. The fast component of the transient decay is more rapid when guanine is located in the second vs first base pair, consistent with an intercalated but not ail end-capped geometry. Spectral broadening Of ultraviolet, fluorescence, and transient absorption spectra is attributed to multiple ground state conformations.