The effects of streptozotocin-induced diabetes mellitus upon mitochondria from rat skeletal muscle and kidney were examined. The rate of amino acid incorporation in vitro by isolated skeletal muscle mitochondria from diabetic animals was decreased by 50–60% from control values. Treatment of diabetic animals with insulin lowered blood glucose levels to control values and restored the rate of muscle mitochondrial protein synthesis in vitro to control levels. The rates of skeletal muscle mitochondrial protein synthesis were also decreased 23–27% by a 2-day fast. Comparison of the translation products synthesized by isolated muscle mitochondria from control and diabetic rats by dodecyl sulfate polyacrylamide-gel electrophoresis revealed a uniform decrease in the synthesis of all polypeptides. Aurintricarboxylic acid and pactamycin, inhibitors of chain initiation, blocked protein synthesis to a greater extent in muscle mitochondria from control as compared to diabetic animals suggesting that mitochondria from diabetics are unable to initiate protein synthesis at a rate comparable to control. Phenotypic changes observed in diabetic muscle mitochondria included a 36% decrease in the content of cytochromes aa 3 and a 27% decrease in cytochrome b, both established as containing mitochondrial translation products in lower eucaryotes. State 3 respiration with glutamate as substrate decreased by 27% and uncoupler-stimulated respiration decreased by 23% in the diabetic mitochondria. By contrast, the specific activities of NADH and succinate dehydrogenases, established as products of cytoplasmic protein synthesis in lower eucaryotes, were not decreased in skeletal muscle mitochondria from the diabetic animals. These results suggest that the considerable muscular atrophy observed in diabetics may involve decreases in both cytoplasmic and mitochondrial protein synthesis, the latter reflected in profound changes in the respiratory chain. By contrast, comparison of kidney mitochondria from control and diabetic rats revealed no differences in the rates of protein synthesis in vitro, nor in the mitochondrial translation products, which corresponded closely to liver and skeletal muscle translation products. Similarly, the mitochondrial content of cytochromes b, c + c 1, and aa 3, the specific activity of succinate dehydrogenase, the rate of state 3 respiration, and the recovery of mitochondria from kidney homogenates did not differ in control and diabetic animals. Kidney mitochondria are thus like liver mitochondria in being relatively unaffected by insulin deprivation.