Aminoacyltransferase II catalyzed the hydrolysis of GTP in the presence of unwashed skeletal muscle ribosomes; however, there was also hydrolysis of the nucleoside triphosphate in the absence of the enzyme. Hydrolysis of GTP in the absence of aminoacyltransferase II was reduced, though not eliminated, by washing the ribosomes in 0.5 M NH4Cl. A further reduction was obtained by adding ATP (0.01 mM). The ribosome dependent aminoacyltransferase II catalyzed hydrolysis of GTP increased linearly for 15 min and was proportional to ribosome concentration; 4–7 moles of the nucleoside triphosphate were hydrolyzed per mole of ribosomes. Ribosomes which had been incubated with puromycin so as to remove peptidyl-tRNA showed no reduction in the aminoacyltransferase II catalyzed hydrolysis of GTP. Ribosomes from the muscle of alloxan-diabetic rats were less active in protein synthesis than those from normal rats. However, there was no difference in the ability of the ribosomes to support GTP hydrolysis even when aminoacyltransferase II was present in saturating amounts. We conclude that the decreased ability of diabetic ribosomes to catalyze protein synthesis is not a result of an inability to utilize aminoacyltransferase II to hydrolyze GTP.