Insulin has been shown to accelerate the incorporation of labeled amino acids into the protein of the isolated rat diaphragm. 1) This insulin stimulation is not exerted through an effect of the hormone on the transport of external amino acids to intracellular sites of protein synthesis. 2) The basic and insulin-enhanced incorporation of radioactive amino acids into protein of isolated cut diaphragm from normal and fasted rats is not affected by the presence or absence of glucose in the incubation medium. 3) Erythrose inhibited simultaneously the basic incorporation of amino acid and its stimulation by insulin. The effect of erythrose is not reversed by Krebs cycle intermediates, but these intermediates permit insulin to enhance protein synthesis, showing that both an intact supply of Krebs cycle intermediates and a glucose utilizing system are neccessary for insulin action. 4) Inhibition of endogenous carbohydrate metabolism in the diaphragm by 2-deoxy-glucose inhibits amino acids incorporation and abolishes the stimulatory effect of insulin. 5) The basic incorporation and its stimulation by insulin are restored to normal by addition of the metabolically active sugars, glucose and mannose. It is concluded that protein synthesis in the isolated diaphragm and its stimulation by insulin are dependent upon some normal glycolitic reaction, but there is some difference of energy supply between the basic incorporation of amino acids and its stimulation by insulin.