AbstractUltraviolet absorption and static fluorescence properties of hexanucleotide (Gm‐A‐A‐Y‐A‐ψp) and a dodecanucleotide (A‐Cm‐U‐Gm‐A‐A‐Y‐A‐ψ‐m5C‐U‐Gp) excised from the anticodon region of phenylalanine tRNA from yeast have been studied with respect to temperature, pH, ionic strength, and Mg2+concentration. At low temperature these oligomers have a largely stacked structure. Only the melting data of the dodecanucleotide in absence of Mg2+fit a two‐state model. From the different melting behavior of the oligonucleotides after excision of base Y, a rodlike structure of the hexanucleotide produced by stacking interactions can be concluded. The Y fluorescence increase produced by Mg2+has been used to evaluate the binding equilibria between Mg2+and the oligonucleotides. One strong binding site per oligonucleotide and a greater number of weak binding sites have been found. The fluorescence of the free base Y is not influenced by Mg2+. The dodecanucleotide enhances the ethidium fluorescence to the same extent as tRNAPheand produces comparable shifts in the excitation and emission spectra. Therefore a double helical structure for this oligomer under the assay conditions is suggested. Only weak binding of ethidium to the hexanucleotide is observed, indicating that intercalation of the dye into its structure is not favored. The data show the decisive role of the nucleobase Y in maintaining a rigid stacked structure of the anticodon nucleotides. This structure is stabilized by high