The topography of the complex of elongation factor G with post-translocative ribosomes has been studied in the Escherichia coli system using fluorescence spectroscopy. We find that a fluorophore attached to the D loop of tRNA is shielded from solvent access by the presence of the factor, and this effect is dependent on factor-promoted GTP hydrolysis. The shielding result suggests that (1) the factor could bind to the tRNA during translocation and (2) the tRNA binding site may be close to that of the factor. The alternative explanation, that the factor affects the conformation of the tRNA bound at a distant site, seems less likely.