The fidelity of protein synthesis depends on specific tRNA aminoacylation by aminoacyl-tRNA synthetase enzymes, which in turn depends on the recognition of the identity of particular nucleotides and structural features in the substrate tRNA. These features generally reside within the acceptor helix, the anticodon stem-loop, and in some systems the variable pocket of the tRNA. In the alanine system, fidelity is ensured by a G.U wobble base pair located at the third position within the acceptor helix of alanine tRNA. We have investigated the activity of mutant alanine tRNAs to explore the mechanism of enzyme recognition. Here we show that the mismatched pair C-C is an excellent substitute for G.U in alanine-tRNA-knockout cells. A structural investigation by NMR spectroscopy of the C-C RNA acceptor end reveals that the two cytosines are intercalated into the helix, and that C-C exists in multiple conformations. Structural heterogeneity also is present in the wild-type G.U RNA, whereas inactive Watson-Crick helices are structurally rigid. The correlation between functional and structural data suggests that the G.U pair provides a distinctive structure and a point of deformability that allow the tRNA acceptor end to fit into the active site of the alanyl-tRNA synthetase. Fidelity is ensured because noncognate and inactive mutant tRNAs are bound in the active site in an incorrect conformation that reduces enzymatic activity.