1. A method is described for the sucrose-gradient sedimentation analysis of ribosomes in a post-mitochondrial supernatant of rat skeletal muscle. 2. An essential feature of the method involves the use of buffer of ionic strength 0.3 for homogenization of the muscle tissue. 3. Polyribosomes can be prepared by precipitation from post-mitochondrial supernatant of skeletal muscle by adjustment of the potassium chloride content of the medium. These polyribosomes stimulate cell-free amino acid incorporation in vitro in an energy-dependent system. 4. Ribosome aggregates of uniform size distribution can be obtained by adjustment of the ionic strength of the post-mitochondrial supernatant, followed by differential sucrose-gradient centrifugation. 5. In vivo, rat skeletal-muscle polyribosomes became labelled by (14)C-labelled amino acid within 15min., and radioactivity was associated with the light ribosome species within 45min. 6. Electron microscopy of the polyribosomes revealed aggregations containing more than 40 single ribosomes.

1. The influence of hydrocortisone, insulin and diet on the size distribution of ribosomes in a post-mitochondrial supernatant prepared from rat skeletal muscle was studied by sedimentation analysis with a linear 15-40% (w/v) sucrose gradient. 2. Within 4hr. after the injection of 5mg. of hydrocortisone to well-nourished rats, a decrease in the yield per g. of muscle and proportion of total RNA due to polyribosomes was observed. Similar results were obtained in rats given a protein-free diet for 3 days before administration of the hormone. 3. Insulin injection increased the yield and proportion of polyribosomes within 2hr. and decreased the proportion of the lighter ribosomal aggregates. Similar results were noted in rats given a protein-free diet for 3 days before injection. A protein-free diet given for 3 days decreased the yield and proportion of polyribosomes. Insulin did not increase the yield of polyribosomes if rats were starved for 52hr. before injection, but decreased the yield and proportion of the lighter ribosome species. 4. A 52hr. period of starvation or 2,4-dinitrophenol (15mg./kg. body wt.) given 1hr. before the rats were killed resulted in a decreased yield and proportion of polyribosomes, and, within 6hr. of re-feeding the rats with protein-free diets, an increased concentration of polyribosomes was noted. 5. The effects of a protein-free diet, hydrocortisone and insulin on the sedimentation of muscle ribosomes were found to be in accord with their net effects on muscle protein synthesis.