Escherichia coli tRNAVal with pyrimidine substitutions for the universally conserved 3′-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNAVal with a 3′-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3′-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNAVal:EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNAVal for EF-Tu:GTP, thus explaining the inability of these tRNAVal variants to function in polypeptide synthesis. Valyl-tRNAVal terminating in 3′-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3′ end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3′ cytosine. Similar observations were made in studies of the interaction of 3′ end mutants of E. coli tRNAAla and tRNAPhe with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3′ end of aminoacyl-tRNAs.