Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tumt) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tumt·GDP complex (KGDP = 1.0 ± 0.1 μM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tumt was used to measure the dissociation constant of the EF-Tumt·GTP complex (KGTP = 18 ± 9 μM). The analysis of these data required information on the dissociation constant of the EF-Tumt·mantGDP complex (KmGDP = 2.0 ± 0.5 μM), which was measured by equilibrium dialysis. Both KGDP and KGTP for EF-Tumt are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tumt·GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tumt. These values are in agreement with a simple equilibrium binding interaction between EF-Tumt and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tumt·GDP complex.