Valine tRNA was purified from 5-fluorouracil (FUra) treated E. coli cells and resolved into two isoaccepting species, termed FUra tRNA(,1)('Val) forms (A) and (B), respectively. The ('19)F NMR spectrum of the (B) form contains 13 resolved resonances for the 14 incorporated FUra residues dispersed from 1.6 and 8.5 ppm (FUra = 0 ppm). ('19)F NMR spectra of the (A) and (B) forms differ in the shift of peak E from ca. 4.3 ppm in form (B) upfield to ca. -15 ppm in form (A). In light of previous sequence studies with FUra tRNA(,F)('Met) and ('19)F spectral differences between the two forms of tRNA(,1)('Val), peak E in the NMR spectrum of FUra tRNA(,1)('Val) (B) is assigned to F17. ('19)F NMR thermal denaturation, bisulfite modification, pH dependence, and Solvent Isotope Shift studies indicate that downfield NMR peaks (4.8-8.5 ppm) correspond to residues participating in tertiary structure interactions, upfield peaks (1.6-3.8 ppm) correspond to residues located in helical domains, and central-field resonances (3.9-4.5 ppm) correspond to residues in relatively unstructured environments. Thermal denaturation studies allow assignment of peak B in the ('19)F NMR spectrum of FUra tRNA(,1)('Val) to F54 and, based on this assignment, demonstrate a thermally-induced low temperature (T(,m) = 36(DEGREES)C) structural transition to a less stacked conformation in the T(psi)C-loop region of the tRNA. Based on a conserved ca. 4 ppm upfield shift of peak A in the ('19)F NMR spectra of FUra tRNA(,1)('Val), tRNA(,f)('Met), and tRNA(,m)('Met) upon removal of Mg('2+), peak A has been tentatively assigned to F55. ('19)F ('19)F nuclear Overhauser effect studies with 6-deutero-FUra tRNA(,1)('Val) agree with and enhance the assignment of peaks A and B to F55 and F54, respectively.;('19)F NMR relaxation parameters were measured and interpreted within the diffusion in a cone or two-state jump formalisms in order to derive motional amplitudes, due to pseudorotational fluctuations of the ribose ring, for the individual 5-fluorouridine (FUrd) residues in FUra tRNA(,1)('Val). These motions occur on the nanosecond time scale and the amplitudes may be correlated directly with the environmental domain of the residue. The large chemical shift anisotropy contributions to the ('19)F linewidths indicate a maintenance of hydrogen-bonding and/or stacking interactions for all FUrd residues in FUra tRNA(,1)('Val). A correlation between residue mobility and solvent exposure is also demonstrated.