Aminoacyl transferase II (T2) is required, together with aminoacyl transferase I (Ti), guanosine triphosphate (GTP), and ribosomes, for the transfer of amino acids from aminoacyl tRNA into protein in the cell-free system from rat liver.'-3 Recent investigations 5 indicate that T2 functions in the elongation of polypeptide chains by catalyzing translocation6' 7 of newly lengthened peptidyl tRNA from an unreactive site on the ribosome to a site in which it again can form a peptide bond. The transferase activity of T2, measured in the presence of T1 by the transfer of amino acids from aminoacyl tRNA into protein, is inhibited by diphtheria toxin and nicotinamide adenine dinucleotide (NAD).8-12 Toxin produces this inhibition by catalyzing the transfer of the ADP ribose moiety of NAD to T2.13 The translocase activity of T2, measured in the absence of T1 by a GTP-dependent increase in the number of polypeptide chains which react with puromycin, is also inhibited by toxin and NAD.5In the present report we describe a ribosome-dependent GTPase activity which cochromatographs on Sephadex G 200 with T2. Recently, the same activity, reminiscent of the ribosome-dependent GTPase in the G factor from E. coli,'4-17 has been observed in both rat liver and rabbit reticulocytes by Felicetti and Lipmann.'8 These authors suggest that the activity has a role in protein synthesis, but they point out that its functional significance has not yet been established. We report that the ribosome-dependent GTPase activity of T2, like its activity measured in the transfer and translocation assays, is inhibited by diphtheria toxin in the presence of NAD. In addition, we show that GTP binds to partially purified T2. In the absence of ribosomes, this binding of GTP to macromolecular material is not influenced by diphtheria toxin and NAD, but inhibition of binding is observed when ribosomes are present. We also present data which show that both GTP and ribosomes protect T2 from toxin-catalyzed ADP ribosylation.Inhibition of the ribosome-dependent GTPase by diphtheria toxin, specifically in the presence of NAD, strongly suggests that the hydrolysis of GTP is related to protein synthesis. These data, together with previous results showing similar inhibitions of T2 activity measured in either the transfer or translocation assay, further suggest that the GTPase reaction is involved in translocation of peptidyl tRNA on the ribosome. Materdals and Methods.-Guanosine-5'-triphosphate-,yP'2 (yP32-GTP) was obtained from International Chemical and Nuclear Corporation; guanosine-5'-triphosphate-H' (Hs-GTP), 1.0 c/mmole, from Schwarz BioResearch, Inc.; and C"-L-phenylalanyl tRNA from E. coli (uniformly labeled), 42 myc/mg, from New England Nuclear Corp.; NAD labeled with H3 in the adenosine moiety (H3-NAD) was prepared as described by 1428