The phenylalanine transfer RNA of baker's yeast (tRNAPhe) contains a base Y of unknown molecular structure next to the anticodon triplet. Since the base Y fluoresces at room temperature (λmax = 431 nm), its emission properties offer a unique tool for studying conformational and binding properties of tRNAPhe. The results obtained by these experiments include the following: (1) The quantum yield of fluorescence of Y in tRNAPhe (φF) is 0.07 ± 0.01 at high Mg2+ concentrations (>10-2 M) and about half that at 10-3 M or less, indicating a [Mg2+]-dependent conformational change of the anticodon loop. (2) The fluorescence of Y isolated from tRNAPhe (Y+) is red-shifted by 15 nm compared to Y in tRNAPhe 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(D2O)/φF(H2O) is 1.5 for tRNAPhe 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 (λmax = 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 Mg2+ 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 × 102 M-1, approximately. A similar complex is formed with poly-uridylate but not with poly-cytidylate. In the absence of Mg2+ the binary complex is not formed.