Unmodified tRNAs are powerful systems to study the effects of posttranscriptional modifications and site-directed mutations on both the structure and function of these ribonucleic acids. To define the general limitations of synthetic constructs as models for native tRNAs, it is necessary to elucidate the conformational states of unmodified tRNAs as a function of solution conditions. Here we report the conformational properties of unmodified yeast tRNAPhe as a function of ionic strength, [Mg2+], and temperature using a combination of spectroscopic measurements along with chemical and enzymatic probes. We find that in low [Na+] buffer at low temperature, native yeast tRNAPhe adopts tertiary structure in the absence of Mg2+. By contrast, tertiary folding of unmodified yeast tRNAPhe has an absolute requirement for Mg2+. Below the melting temperature of the cloverleaf, unmodified yeast tRNAPhe exists in a Mg2+-dependent equilibrium between secondary and tertiary structure. Taken together, our findings suggest that although the tertiary structures of tRNAs are broadly comparable, the intrinsic stability of the tertiary fold, the conformational properties of intermediate states, and the stability of intermediate states can differ significantly between tRNA sequences. Thus, the use of unmodified tRNAs as models for native constructs can have significant limitations. Broad conclusions regarding "tRNA folding" as a whole must be viewed cautiously, particularly in cases where structural changes occur, such as during protein synthesis.