We have studied the interactions between Escherichia coli tRNAVal and valyl-tRNA synthetase (ValRS) by enzymatic footprinting with nuclease S1 and ribonuclease V1, and by analysis of the aminoacylation kinetics of mutant tRNAVal transcripts. Valyl-tRNA synthetase specifically protects the anticodon loop, the 3' side of the stacked T-stem/acceptor-stem helix, and the 5' side of the anticodon stem of tRNAVal against cleavage by double- and single-strand-specific nucleases. Increased nuclease susceptibility at the ends of the anticodon- and T-stems in the tRNAVal.ValRS complex is indicative of enzyme-induced conformational changes in the tRNA. The most important synthetase recognition determinants are the middle and 3' anticodon nucleotides (A35 and C36, respectively); G20, in the variable pocket, and G45, in the tRNA central core, are minor recognition elements. The discriminator base, position 73, and the anticodon stem also are recognized by ValRS. Replacing wild-type A73 with G73 reduces the aminoacylation efficiency more than 40-fold. However, the C73 and U73 mutants remain good substrates for ValRS, suggesting that guanosine at position 73 acts as a negative determinant. The amino acid acceptor arm of tRNAVal contains no other synthetase recognition nucleotides, but regular A-type RNA helix geometry in the acceptor stem is essential [Liu, M., et al. (1997) Nucleic Acids Res. 25, 4883-4890]. In the anticodon stem, converting the U29:A41 base pair to C29:G41 reduces the aminoacylation efficiency 50-fold. This is apparently due to the rigidity of the anticodon stem caused by the presence of five consecutive C:G base pairs, since the A29:U41 mutant is readily aminoacylated. Identity switch experiments provide additional evidence for a role of the anticodon stem in synthetase recognition. The valine recognition determinants, A35, C36, A73, G20, G45, and a regular A-RNA acceptor helix are insufficient to transform E. coli tRNAPhe into an effective valine acceptor. Replacing the anticodon stem of tRNAPhe with that of tRNAVal, however, converts the tRNA into a good substrate for ValRS. These experiments confirm G45 as a minor ValRS recognition element.