15N-Labeled Bacillus subtilis tRNA(Trp) wild type and a series of mutants were hyperexpressed in Escherichia coli and purified for NMR studies with the use of two-dimensional nuclear Overhauser effect spectroscopy (NOESY) and heteronuclear single quantum correlation (HSQC) and three-dimensional NOESY-HSQC techniques. These made possible chemical shift assignments of imino protons and determination of the thermal stability of the tRNA(Trp) molecules. Almost all of the imino protons in the helical regions and the tertiary base pairs were assigned, except three imino protons of the AU base pairs whose peaks were not clearly observed. Several base triplets found in the crystal structure of tRNA were observed in the present study as well. These studies also revealed two components of tRNA(Trp), which could not be separated by high pressure liquid chromatography, corresponding to s(4)U and U at position 8 of the tRNA(Trp), as indicated by two different sets of peaks for the TpsiC and D arms. The modification at position 8 altered the local conformation of the core region of the tRNA. Thermal unfolding experiments showed that the unfolding process is cooperative in the presence of a high concentration of magnesium ions and that the component corresponding to the s(4)U8 is more stable than the U8 component, thus providing evidence that the thiolation of U8 stabilizes the tertiary structure of tRNA.