Tetracycline (Tc) is a broad-spectrum antibiotic that kills bacteria by interrupting protein biosynthesis. It is thought that the bacteriostatic action of Tc is associated with its binding to the acceptor site (or A site) in the bacterial ribosome, interfering with the attachment of aminoacyl-tRNA. Recently, however, the crystal structure of a complex between Tc and trypsin-modified elongation factor Tu (tm-EF-Tu) was determined, raising the question of whether Tc binding to EF-Tu has a role in its inhibition of protein synthesis. We address this question using computer simulations. As controls, we first compute relative ribosome binding free energies for seven Tc variants for which experimental data are available, obtaining good agreement. We then consider the binding of Tc to both the trypsin-modified and unmodified EF-Tu-GDP complexes. We show that the direct contribution of EF-Tu to the binding free energy is negligible; rather, the binding can be solely attributed to interactions of Tc with a bridging Mg(2+) ion and the GDP phosphate groups. The effects of trypsin modification are modest. Further, our calculations show that EF-Tu does not exhibit any binding preference for Tc over the nonantibiotic, 4-dedimethyl-Tc, and EF-Tu does not bind the Tc analogue tigecycline, which is a potent antibiotic. In contrast, both the ribosome and the Tet Repressor protein (involved in Tc resistance) do show a binding preference for Tc over 4-dedimethyl-Tc, and the ribosome prefers to bind tigecycline over Tc. Overall, our results provide insights into the binding properties of tetracyclines and support the idea that EF-Tu is not their primary target.