Certain aminoacyl-tRNA synthetases discriminate between closely similar amino acids by hydrolytic editing reactions in the presence of their cognate tRNA. An example is the class I isoleucyl-tRNA synthetase. We recently showed that a mutation which eliminates discrimination between isoleucine (Ile) and valine (Val) in the initial amino acid binding and activation steps had little effect on the hydrolytic editing of activated valine in the presence of isoleucine tRNA (tRNA(Ile)). The results showed that initial amino acid binding and discrimination are functionally independent of tRNA-dependent amino acid discrimination. In this work, we cross-linked (to isoleucyl-tRNA synthetase) a reactive analog of valine misacylated onto tRNA(Ile). Mutation of specific residues within a peptide segment identified by the cross-linking analysis severely affected discrimination of Val-tRNA(Ile) versus Ile-tRNA(Ile). The mutationally sensitive residues are part of an insertion into the catalytic domain and are themselves completely conserved among all known prokaryotic and eukaryotic sequences of the enzyme.