The effect of guanylyl-5′-methylene diphosphonate (GMD) on the binding of Phe-tRNA to the acceptor site of a ribosome·poly (U)·AcPhe-tRNA complex and on the subsequent incorporation of the Phe-tRNA into peptide linkage has been studied. The binding that occurs in the presence of TIs, TIu, and GMD differs in several respects not only from that obtained in the presence of TIs, TIu, and GTP but also from that obtained in the absence of TIs and TIu, i.e., nonenzymatically. Nonenzymatic binding of Phe-tRNA to the acceptor site of the ribosome·poly (U)·AcPhe-tRNA complex occurs at high concentrations of Mg2+ (8–20 mM) and a significant portion of the Phe-tRNA interacts with AcPhe-tRNA to form AcPhe-Phe-tRNA. In the presence of TIs, TIu, and GTP, maximal binding of Phe-tRNA to the ribosomal complex occurs at low concentrations of Mg2+ (4–6 mM); the GTP is hydrolyzed; TIu·GDP, and Pi are released from the ribosome; and most of the Phe-tRNA bound to the ribosomes interacts with the AcPhe-tRNA to form dipeptide. In contrast, the concentration of Mg2+ required to obtain maximal binding of Phe-tRNA to the ribosomes in the presence of TIs, TIu, and GMD is about 12 mM; the TIu and GMD remain bound to the ribosomes; and very little dipeptide is formed. Moreover, the binding of Phe-tRNA to the ribosomes in the presence of GMD and transfer factors is very unstable. These data indicate that the presence of TIu and GMD on the ribosomes prevents the interaction of the Phe-tRNA at the acceptor site with the AcPhe-tRNA at the donor site, and suggest that hydrolysis of GTP and release of TIu and GDP from the ribosomes are prerequisites for peptide bond formation. In addition, evidence is presented which demonstrates that Phe-tRNA bound to the ribosomes in the presence of GMD is not incorporated into dipeptide upon the subsequent addition of GTP, indicating that the GMD bound to the ribosomes does not exchange with GTP.