Elongation factor Tu (EF-Tu), in the presence of Phe-tRNA, GMPPCP, and Poly (U), binds to 70S ribosomes at the recognition (R) site. In order to identify the ribosomal proteins adjacent to the EF-Tu occupying the R site, EF-Tu:Phe-tRNA:GMPPCP:ribosome complexes were crosslinked by modification with 2-iminothiolane and mild oxidation to form disulfide bridges between neighbouring proteins whose endogenous or introduced SH groups were appropriately located. The binding of Phe-tRNA to the ribosome was shown to be largely dependent on the presence of Poly(U). The total protein from the complexes was extracted and separated by two-dimensional gel electrophoresis by non-equilibrium pH gradient electrophoresis (NEpHGE) in the first dimension, followed by gradient SDS gel electrophoresis in the second dimension. Comparison of control samples crosslinked without Poly(U) to those crosslinked with Poly(U) present showed a single crosslinked complex in the region of the gel near EF-Tu. No cross-links in the vicinity of EF-Tu were visible in the absence of Poly(U). The crosslinked proteins in this region were recovered by electroelution, radiolabeled and their identity was confirmed by 2D gel electrophoresis and immunoblot analyses. Two major 50S ribosomal proteins, L7/L12 and L10 were found to be covalently linked to EF-Tu. The isolated crosslinked complex did not contain any protein from the 30S subunit. These results demonstrate that L7/L12 and L10 are the major, if not only, ribosomal protein cross-links to EF-Tu in the R site. In contrast to previous crosslinking results obtained by others, our results define a unique location for the EF-Tu binding site, one compatible with functional data and near that of the EF-G binding site on the ribosome.