The involvement of tRNA structural elements beyond the anticodon in aminoacyl-tRNA (aa-tRNA) selection by the ribosome has revealed that substrate recognition is considerably more complex than originally envisioned in the adaptor hypothesis. By combining recent breakthroughs in aa-tRNA synthesis and mechanistic and structural studies of protein synthesis, we have investigated whether aa-tRNA recognition further extends to the amino acid, which would explain various translation disorders exhibited by misacylated tRNAs. Contrary to expectation, we find that natural amino acids misacylated onto natural but non-native tRNAs are selected with efficiencies very similar to those of their correctly acylated counterparts. Despite this, small but reproducible differences in selection indeed demonstrate that the translational machinery is sensitive to the amino acid-tRNA pairing. These results suggest either that the ribosome is an exquisite sensor of natural versus unnatural amino acid-tRNA pairings and/or that aa-tRNA selection is not the primary step governing the amino acid specificity of the ribosome.