This laboratory project is one component of a semester-long advanced biochemistry laboratory course that uses several complementary techniques to study tRNA[superscript Phe] conformational changes induced by ligand binding. In this article we describe a set of experiments in which students use fluorescence spectroscopy to study tRNA[superscript Phe] structure. Both intrinsic (Y Base) and extrinsic (ethidium bromide) fluorescence probes are monitored. Ligands (e.g., spermine, neomycin B, magnesium) are titrated into tRNA[superscript Phe] solution and changes in fluorescence intensity are monitored. Students fit the data to hyperbolic saturation curves to determine ligand-binding equilibrium constants and draw conclusions on how different binding modes affect tRNA[superscript Phe] tertiary structure. (Contains 3 tables, 1 figure and 2 notes.)