To test the hypothesis that cortisol and epinephrine have direct regulatory roles in muscle glycogen metabolism and to determine what those roles might be, we developed an in vitro white muscle slice preparation from rainbow trout. In the absence of hormones, glycogen-depleted muscle slices obtained from exercised trout were capable of significant glycogen synthesis and the amount of glycogen synthesized was inversely correlated with post-exercise glycogen content. When post-exercise glycogen levels were < 5 µmol.g^-1, about 4.3 µmol .g^-1 of glycogen was synthesized, but when post-exercise glycogen levels were >5 µmol .g^-1, only about 1.7 µmol .g^-1 of glycogen was synthesized. This difference in the amount of glycogen synthesized was reflected in the degree of activation of glycogen synthase. Post-exercise glycogen content also influenced the response of the muscle to 10^-8M epinephrine and 10^-8M dexamethasone (a glucocorticoid analogue). At high glycogen levels (>5 µmol .g^-1), epinephrine and dexamethasone stimulated glycogen phosphorylase activity and net glycogenolysis, whereas at low (<5 µmol.g^-1) glycogen levels, glycogenesis and activation of glycogen synthase activity prevailed. These data clearly indicate not only is trout muscle capable of in situ glycogenesis, but the amount of glycogen synthesized is a function of initial glycogen content. Furthermore, while dexamtheasone and epinephrine directly stimulate muscle glycogen metabolism, the net effect is dependent upon initial glycogen content.