The interaction of the polypeptide chain elongation factor G (EF-G) from E. coli with guanine nucleotides was investigated using the hydrophobic dye, 1-anilino-8-naphthalensulfonic acid. It was found that the fluorescence intensity of the hydrophobic dye elicited in the presence of EF-G was diminished markedly by addition of GTP, and to a lesser extent, by addition of GDP. Direct evidence for the formation of the binary complexes, EF-G-GTP and EF-G-GDP, was provided by gel filtrations of EF-G on Sephadex G-25 columns equilibrated with buffers containing radioactive GTP and GDP, respectively.