A DNA hairpin containing a T3 loop, as occurs in the terminal repeat of a popular gene therapy vector (Adenoassociated Virus 2, AAV2), has been extensively studied using homo- and heteronuclear NMR experiments. Almost complete assignment of the proton and carbon resonances, including H5'(Pro-S) and H5'(Pro-R) protons, has been accomplished at natural abundance. NOESY spectra in H2O and D2O have revealed many unusual NOEs, which, when combined with the epsilon, beta, gamma, and chi torsion angles determined from heteronuclear 1H-13C, 1H-31P, and 13C-31P coupling constants, have allowed for a more detailed picture of the T3 mini-loop hairpin. The three loop thymidines are all unpaired, yet are highly structured when bracketed by a 5'-GC...GC-3' stem sequence. The structure determined in this manuscript is considerably different from several other structures reported so far. Contrary to an RNA oligomer with a central U3 sequence that has the tendency to form a duplex with three U*U mismatches, the d(GAAGC-TTT-GCTTC) sequence exists mostly as a hairpin under millimolar NMR conditions. Since T3 triloop was found to be an essential element for the site-specific non-homologous integration of the AAV2 virus, and modification of the T3 loop residue abolishes such capability, the structure we report here may be of biological significance.