Escherichia coli cysteinyl-tRNA synthetase (CysRS) achieves a high level of amino acid specificity without an editing reaction. The crystal structure of CysRS bound to substrate cysteine suggested that direct thiol coordination to a tightly bound zinc ion at the base of the active site is the primary determinant of selectivity against non-cognate amino acids. This hypothesis has now been supported by spectroscopic studies of cobalt-substituted CysRS. Binding of cysteine, but not non-cognate amino acids, induces high absorption in the ligand-to-metal charge transfer region, providing evidence for formation of a metal-thiolate bond. In addition, mutations in the zinc ligands alter the absorption spectrum without reducing the discrimination against non-cognate amino acids. These results argue strongly for a major role for the zinc ion in amino acid discrimination by CysRS, where the tight zinc-thiolate interaction and the strict structural geometry of the metal ion are sufficient to reject serine by more than 20,000-fold at the binding step.