The interactions among Escherichia coli elongation factor G (EF-G), guanine nucleotides, ribosomes, and fusidic acid were investigated by a number of physical techniques. Equilibrium dialysis studies demonstrated the existence of a binary EF-G-GDP complex. This complex forms with a stoichiometry of ca 1:1 and an apparent Ka of 2.5 X 10(5) M-1. While no evidence was obtained for the formation of a ribosome-GDP complex, in the presence of ribosomes, the apparent Ka for guanosine diphosphate (GDP) increased 40-fold over that for binding to EF-G alone. Although the apparent Ka increased, the stoichiometry remained ca. 1 mol of GDP/mol of EF-G. An upper limit of 1.3 X 10(7) M-1 was calculated for the Ka for binding of ribosomes to the EF-G-GDP complex. Fusidic acid had no effect on the apparent Ka's for either the EF-G-GDP or EF-G-beta, gamma-methyleneguanosine triphosphate (GMP-P(CH2)P)=ribosome complexes, but markedly increased the Ka for GDP in the EF-G-GDP-ribosome complex without altering the stoichiometry. The apparent Ka for GDP was shown to be dependent upon the fusidic acid concentration. In addition, the rate of GDP exchange into the quaternary EF-G-GDP-ribosome-fusidic acid complex was inversely related to the fusidic acid concentration. All of the data obtained in these studies suggest that the formation and dissociation of complexes involving EF-G and guanine nucleotides is ordered. GDP is the first component to bind to EF-G, followed by the ribosome, and, finally, fusidic acid. This conclusion is consistent with the kinetic mechanism for the hydrolysis of GTP by EF-G and the ribosome proposed in the preceding paper of this issue (Rohrbach and Bodley (1976b). In addition to these binding studies, guanine nucleotides have also been shown to protect EF-G against both limited trypsinolysis and chemical modification by N-ethylmaleimide. These observations offer additional evidence for the existence of a guanine nucleotide binding site on EF-G.