('19)F NMR was used to study E. coli tRNA(,1)('Val), tRNA(,f)('Met), and tRNA(,m)('Met), in which 5-fluorouracil (FUra) has replaced uracil and uracil-derived minor bases. ('19)F NMR spectra of these tRNAs resolve resonances from nearly all the incorporated FUra residues. Each of the three tRNAs can be resolved into two isoaccepting species, termed forms A and B, whose ('19)F spectra differ in the shift of one ('19)F peak from ca. 4.5 ppm in form B, upfield to -15 ppm in form A. Because the two isoacceptors of each tRNA differ only at one position, the peaks at 4.5 ppm in the spectra of (FUra)tRNA(,1)('Val) and (FUra)tRNA(,m)('Met) are assigned to FUra 17 and Fura 20 respectively.;Bisulfate modification and pH dependence indicate that ('19)F signals in the central region of the spectrum of (FUra)tRNA(,1)('Val) correspond to fluorouracils in non-base-paired regions. Photoreaction with psoralen indicates upfield ('19)F signals arise from residues in helical environments. Removal of magnesium or addition of NaCl produces major, reversible changes in the ('19)F spectrum of fluorinated tRNAs. Studies of manganese and spermine binding to (FUra)tRNA(,1)('Val) allow localization of several resonances in the ('19)F spectrum to regions near putative binding sites for these ions.;Binding of the codon G(,p)U(,p)A causes an upfield shift of a ('19)F resonance at 3.9 ppm in the spectrum of (FUra)tRNA(,1)('Val). G(,p)U(,p)A(,p)A, which is complementary to the anticodon and 5'-adjacent FUra 33, shifts an additional ('19)F peak at 4.5 ppm. ('1)H NMR and RNase H digestion studies show that the oligonucleotides bind to the anticodon. The shift of the ('19)F peak at 4.5 ppm is specific for G(,p)U(,p)A-containing tetranucleotides having a 3'-terminal adenosine, supporting the possibility of anticodon loop conformation different from that in the crystal structure. Based on these studies, the ('19)F peaks at 3.9 and 4.5 ppm can be assigned to FUra 34 and 33 in (FUra)tRNA(,1)('Val). Magnesium and spermine were shown to influence the codon-anticodon interaction. The codon methionine A(,p)U(,p)G also shifts a peak in the central part of the ('19)F spectrum of (FUra)tRNA(,m)('Met). However, A(,p)U(,p)G has no effect on the spectrum of (FUra)tRNA(,f)('Met), indicating possible differences between the anticodon loop of elongator and initiator tRNAs. ('19)F NMR studies detect no binding of C(,p)G(,p)A(,p)A to the T loop of (FUra)tRNA(,1)('Val), in the absence or presence of codon, indicating the tertiary interactions between the T and D loops are not disrupted by codon binding.