The phenylalanine transfer RNA of baker's yeast (tRNA Phe ) contains a base Y of unknown molecular structure next to the anticodon triplet. Since the base Y fluoresces at room temperature (λ max = 431 nm), its emission properties offer a unique tool for studying conformational and binding properties of tRNA Phe . The results obtained by these experiments include the following: ( 1 ) The quantum yield of fluorescence of Y in tRNA Phe (φF) is 0.07 ± 0.01 at high Mg 2+ concentrations (>10 -2 M ) and about half that at 10 -3 M or less, indicating a [Mg 2+ ]-dependent conformational change of the anticodon loop. ( 2 ) The fluorescence of Y isolated from tRNA Phe (Y + ) is red-shifted by 15 nm compared to Y in tRNA Phe which suggests a stacked (more hydrophobic) environment for Y in the intact anticodon loop. φF of Y + is 0.035. ( 3 ) The solvent isotope effect φF(D 2 O)/φF(H 2 O) is 1.5 for tRNA Phe and 1.9 for Y + i.e., Y in tRNA is still hydrated. ( 4 ) The temperature dependence of φF in a polar glass shows that quenching occurs only at temperatures at which the glass has sufficiently low viscosity to permit solvent shell relaxation in the excited state. The low-temperature (80°K) fluorescence is blue shifted (λ max = 409 nm) and the phosphorescence has a decay time of 1.5 seconds, a threshold at 392 nm and a spectral shape like that of guanine. ( 5 ) In the presence of 10 -2 M Mg 2+ penta-uridylate, which contains the codon triplet, a small blue shift and a decrease in φF are observed. This shift can be used to establish the formation of a binary complex between the codon and the anticodon with an association constant of 4 × 10 2 M -1 , approximately. A similar complex is formed with poly-uridylate but not with poly-cytidylate. In the absence of Mg 2+ the binary complex is not formed.