Denervation has been shown to impair the ability of insulin to stimulate glycogen synthesis and to a lesser extent glucose transport in rat skeletal muscle. Insulin binding to its receptor, activation of the receptor tyrosine-kinase and phosphatidylinositol 3'-kinase do not appear to be involved. On the other hand, it has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity. In this paper, we characterize further these changes in PKC and assess other possible signaling abnormalities that might be related to the decrease of glycogen synthesis. The results reveal that PKC and , but not , , are increased in the membrane fraction 24 h after denervation, and that the timing of these changes parallel the impaired ability of insulin to stimulate glycogen synthesis. At 24 hr these changes were associated with a 65% decrease in glycogen synthase (GS) activity ratio and decreased electrophoretic mobility indicative of phosphorylation in GS in muscles incubated in the absence of insulin. Incubation of the denervated soleus with insulin for 30 min minimally increased glucose incorporation into glycogen; however, it increased GS activity 3-fold, to a value still less that of control muscle, and it eliminated the gel shift. In addition, insulin increased the apparent abundance of GSK3 and PP1 in the supernatant fraction of muscle homogenate to control values and it caused the same increases in GSK3 and Akt/PKB phosphorylation and Akt/PKB activity that it did in non-denervated muscle. No alterations in hexokinase I or II activity were observed after denervation; however, in agreement with a previous report glucose-6-phosphate levels were diminished in 24 h denervated soleus and they did not increase after insulin stimulation. These results indicate that, alterations in the distribution of PKC and accompany the impairment of glycogen synthesis in the 24 h denervation soleus. They also indicate that the basal rate of glycogen synthesis and its stimulation by insulin in these muscles are diminished despite a normal activation of Akt/PKB and phosphorylation of GSK3. The significance of the observed alterations to GSK3 and PP1 distribution remain to be determined.