Abstract: In the present investigations the molecular unfolding of yeast tRNA(Gly) has been studied by a combination of nuclear magnetic resonance spectroscopy, melting techniques, and relaxation kinetics. From these studies the following pathway of unfolding was found. In a coupled melting transition the tertiary, the DHU, and the anticodon structure are disrupted. This is followed by the melting of the acceptor arm, while the T psi C arm, which only contains G-C pairs, melts out last. Interestingly, during the first melting transition a new structure not belonging to the original cloverleaf structure is formed. The thermodynamic and kinetic parameters of the melting transitions were determined and are discussed in relation to earlier work. The present nuclear magnetic resonance (NMR) experiments as well as earlier studies show that the ring current calculations based on the cloverleaf structure provide a good first-order interpretation of the NMR spectra of tRNA.