Although the structural basis for amino acid activation by class I tRNA synthetases is known, that for their editing activities has remained elusive. Two class I tRNA synthetases discriminate closely similar amino acids by RNA-independent and RNA-dependent mechanisms. In the absence of tRNA, isoleucyl-tRNA synthetase misactivates valine, while valyl-tRNA synthetase misactivates threonine. Both enzymes improve amino acid discrimination by tRNA-dependent hydrolytic editing reactions. Recent mutational analysis of an isoleucyl-tRNA synthetase showed that discrimination of valine from isoleucine by amino acid activation was functionally independent of discrimination by editing. In this work, we used mutational analysis to test whether the two types of amino acid discrimination were functionally independent in valyl-tRNA synthetase. We obtained four mutations in the valine enzyme which severely affected amino acid activation. The two most defective enzymes reduced kcat/Km for activation of valine by more than 4 orders of magnitude and were essentially inactive for aminoacylation. These two defective enzymes were tested and found to be unaltered in catalysis of rapid and selective removal of threonine misacylated onto valine tRNA. On the basis of these data, and in spite of there being few residues conserved between the two proteins in a region believed important for editing, we propose that the valine and isoleucine enzymes share a global design which functionally separates amino acid editing from amino acid activation.