The hydrodynamics of aminoacyl-tRNA free and bound to protein elongation factor Tu (EF-Tu) were examined using multifrequency phase and modulation fluorometry. The probes fluorescein, covalently linked to Phe-tRNAphe (Phe-tRNAphe-F8), and ethidium bromide, noncovalently bound to Phe-tRNAphe, were utilized. At 5°C the Debye rotational relaxation times found for ethidium bromide bound to tRNA and to the complex, EF-TuPhe-tRNAphe, were 136 ns and 195 ns, respectively. Both values are longer than would be expected for spheres of equal volume and reflect the irregular shapes of the tRNA and ternary complexes. Global rotational relaxation times (5°C) for Phe-tRNAphe-F8 free and bound to EF-TuGTP were 103 ns and 206 ns, respectively, consistent with the ethidium bromide results. A second, faster rotational relaxation time of approximately 4.7 ns, presumably due to local structural motion of the fluorescein, was unchanged upon ternary complex formation. This result suggests that the local mobility of the bound fluorescein is not hindered by the presence of the protein, and it is therefore unlikely that the region of the tRNA which includes the fluorescein-labeled residue S4-U-8, interacts closely with EF-Tu in the ternary complex. The dynamic polarization measurements on the ethidium bromide/tRNA system also demonstrated the interesting phenomenon of anomalous phase delays, an effect which is quite sensitive to small amounts of free or bound probe.