Elongation factor Tu (EF-Tu) binds all elongator aminoacyl-transfer RNAs (aa-tRNAs) for delivery to the ribosome during protein synthesis. Here, we show that EF-Tu binds misacylated tRNAs over a much wider range of affinities than it binds the corresponding correctly acylated tRNAs, suggesting that the protein exhibits considerable specificity for both the amino acid side chain and the tRNA body. The thermodynamic contributions of the amino acid and the tRNA body to the overall binding affinity are independent of each other and compensate for one another when the tRNAs are correctly acylated. Because certain misacylated tRNAs bind EF-Tu significantly more strongly or weakly than cognate aa-tRNAs, EF-Tu may contribute to translational accuracy.