The structural basis for the recognition of valine transfer RNA (tRNA[superscript] Val) by valyl-tRNA synthetase (VRS) from E. coli has been investigated by [superscript]19F NMR spectroscopy, aminoacylation kinetics and the technique or site-directed mutagenesis. The accomplishments presented here entail (1) the development of protocols for the synthesis of wild type and mutant tRNA[superscript] Val having 5-fluorouracil in place of uracil, (2) the direct assignment of [superscript]19F NMR peaks to specific FUra residues in tRNA[superscript] Val, and (3) the role played by specific bases in the recognition of tRNA[superscript] Val by its cognate synthetase;For in vitro synthesis of FUra-substituted tRNA[superscript] Val, plasmid pVAL119-21, combining a synthetic E. coli tRNA[superscript] Val gene, along with the T7 RNA promoter, was constructed. The synthetic tRNA gene has incorporated at its 3[superscript]' terminus the restriction site for BstN1. Runoff transcription by T7 RNA polymerase of the BstN1 digested plasmid yields tRNA[superscript] Val which has no minor bases. The tRNA transcript, purified by HPLC, has a valine acceptance activity of 1200-1300 pmole/A[subscript]260;The [superscript]19F NMR spectra of the transcribed and native (FUra)tRNA[superscript] Val are identical except for slight differences in the chemical shifts of resonances in the central region of the spectrum (peaks E and F). Comparison of [superscript]19F NMR spectra of wild type and mutant forms of (FUra)tRNA[superscript] Val leads to the assignment of peaks B, C, E, F, H, K and L to fluorouracil 64, 59, 47, 33, 34, 29 and 67 respectively;Aminoacylation kinetic studies show that most of the mutant tRNAs could be aminoacylated by VRS. However, replacement of C36 with A, G, or U results in 150-1000 fold decrease in V[subscript] max/K[subscript] m. Mutants C34 and G41 also have lower V[subscript] max/K[subscript] m values. Addition of VRS induces several changes in the [superscript]19F NMR spectrum of (FUra) tRNA[superscript] Val. Intensity at 5 ppm increases, while the signal at 4 ppm (peak G/H) loses intensity. One peak shifts downfield from the cluster around 3 ppm. These results suggest an involvement of the anticodon in the recognition of tRNA[superscript] Val by VRS.