In this paper we unveil a new homogeneous assayusing TOTO-3 and the polythymine oligonucleotide T 33for the highly selective and sensitive detection of Hg (2+) in aqueous solution. The fluorescence of TOTO-3 is weak in the absence or presence of randomly coiled T 33. After T 33 interacts specifically with Hg (2+) ions through T-Hg (2+)-T bonding, however, its conformation changes to form a folded structure that preferably binds to TOTO-3. As a result, the fluorescence of a mixture of T 33 and TOTO-3 increases in the presence of Hg (2+). Our data from fluorescence polarization spectroscopy, capillary electrophoresis with laser-induced fluorescence detection, circular dichroism spectroscopy, and melting temperature measurements confirm the formation of folded T 33-Hg (2+) complexes. Under optimum conditions, the TOTO-3/T 33 probe exhibited a high selectivity (>or=265-fold) toward Hg (2+) over other metal ions, with a limit of detection of 0.6 ppb. We demonstrate the practicality of this TOTO-3/T 33 probe for the rapid determination of Hg (2+) levels in pond water and in batteries. This approach offers several advantages, including rapidity (<15 min), simplicity (label-free), and low cost.