Enhanced luminescence resulting from energy transfer (EnT) from nucleic acids to Tb3+ has been utilized to investigate the binding of the ions to the bases and nucleotides, as well as in the detection of single mismatches in duplexes. Cytosine enhances the Tb3+ emission, but dCMP does not, indicating that the lanthanide bound to the phosphate group is too far away from the base for efficient energy transfer. Conversely, the enhancement of the Tb3+ luminescence by dGMP is greater than that of G, where the phosphate appears to aid in the binding of the ion to the base. We propose that the phosphate group in dGMP is able to fold over and permit coordination of the ion to the O6 and N7 atoms of the base while still bound to the anionic phosphate oxygens, thus increasing the binding affinity and promoting efficient EnT. Single-stranded oligonucleotides greatly enhance the Tb3+ emission, but duplexes do not. Single mismatches in the sequence of a duplex lead to selective luminescence enhancement in the presence of Tb3+. Th, largest enhancement was observed for the GG mismatch, followed by CA, GA, and CC, and the smallest emission intensity was measured for TT and TG mismatches. The unexpected role of adenine in the emission enhancement has been explained through preassociation of the Tb3+, thus permitting A to be in the coordination sphere of the ion. It was concluded that A is able to transfer energy to Tb3+ when bound to the ion, but in the absence of the supramolecular assembly, it cannot coordinate strongly enough to the lanthanide to effect EnT. The low emission enhancement by the TG mismatch has been explained in terms wobble pair formation. These findings show that the enhanced emission of lanthanides can be successfully utilized to selectively detect single mismatches in duplexes.