1. 1. The role of adenosine 3′,5′-phosphate (cyclic AMP) in the mechanism of action of insulin on the enzyme glycogen transferase (UDP glucose: α-1,4-glucan α-4-glucosyltransferase, EC 2.4.1.11) was investigated in rat diaphragm muscle. 2. 2. Incubation of intact rat diaphragms with insulin alone did not alter the cyclic AMP content of the muscle. When the diaphragms were exposed to both insulin and epinephrine simultaneously, the increase in the cyclic AMP concentration was the same as with epinephrine alone. Under these conditions, insulin produced the previously observed increase in the proportion of glycogen transferase in the glucose-6-phosphate-independent (I) form, but had no significant effect on glycogen phosphorylase (α-1,4-glucan: orthophosphate glucosyltransferase, EC 2.4.1.1). 3. 3. When diaphragms were pe-incubated with insulin, there was a diminished rise in cyclic AMP levels in the first few minutes after the addition of epinephrine. An increase in activity of the I form of glycogen transferase was observed, but even under these conditions, phosphorylase activity was not altered significantly by insulin. 4. 4. These results suggest that the effect of insulin on glycogen transferase activity in skeletal muscle does not depend upon changes in the tissue concentration of cyclic AMP. © 1969.