Hydrogen exchange rates of the imino protons of the thrombin-binding 15 mer DNA aptamer d(G(1)G(2)T(3)T(4)G(5)G(6)T(7)G(8)T(9)G(10)G(11)T(12)T(13)G(14)G(15)) in the presence of Sr(2+) were measured. In the temperature range 15-35 degrees C, the exchange rates of the eight iminos in the quadruplex core were not uniform, with the G(2), G(11) and G(15) iminos exchanging faster, the G(1), G(5), G(10) and G(14) iminos exchanging slower, and the G(6) imino exchanging at a medium rate. In the quadruplex G(1), G(5), G(10) and G(14) adopted syn glycosidic conformation, while G(2), G(6), G(11) and G(15) adopted anti-conformation. It was found that the four slowly exchanging iminos, which were all the syn-iminos, happened to be located in the TT loops that were not easy to open to the solvent. The anti-iminos exchanged faster, but the G(6) imino exchanged slower than other anti-iminos, because its hydrogen bond with the G(10)O6 was stabilized by the TGT loop. The fact that the G(6) imino exchanged at a faster rate than those syn-iminos in the TT loops suggested that the TGT loop was less stable than the TT loops. Unfolding mechanism for the quadruplex was thus proposed: The quadruplex first uncoupled the three base pairs: G(1)-G(15), G(2)-G(14) and G(5)-G(11), which were not protected by any loops. Then it opened the TGT loop. Finally, it opened the TT loops and the sequence became an unstructured random coil that exchanged with the quadruplex conformation. The conformational exchange between the quadruplex and random coil had been detected.