The complex of Escherichia coli elongation factor Tu with GTP (EF-Tu.cntdot. GTP) reacts with N.epsilon.-bromoacetyl-Lys-tRNA (.epsilon.BrAcLys-tRNA) to form a functional covalently linked complex (XLTC). The site of cross-linking must be near the site on EF-Tu.cntdot.GTP that binds the aminoacyl moiety of aminoacyl (AA) tRNA. For identification of this site, 1 nmol of purified XLTC prepared from .epsilon.BrAc[14C]Lys-tRNA was digested first with RNase A and then with trypsin, and the peptides were resolved by high-performance liquid chromatography using a C8 reverse-phase column. A single peptide contained 80% of the label. The amino acid composition of this peptide was identical with that of residues 59-74 in EF-Tu. The NH2-terminal sequence of the peptide was determined to be Gly-Ile-Thr-Ile, which are residues 59-62 in EF-Tu. The modified amino acid was identified as .pi.-(carboxymethyl)histidine, which establishes that His-66 is at or near the AA-tRNA binding site on EF-Tu.cntdot.GTP.