To determine whether prior exercise affects the pathways of liver and muscle glycogen synthesis, rested and postexercised rats fasted for 24 h were infused with glucose (200 mumol.min-1.kg-1 iv) containing [6-3H]glucose. Hyperglycemia was exaggerated in postexercised rats, but blood lactate levels were lower than in nonexercised rats. The percent of hepatic glycogen synthesized from the indirect pathway (via gluconeogenesis) did not differ between exercised (39%) and nonexercised (36%) rats. In red muscle, glycogen was synthesized entirely by the direct pathway (uptake and phosphorylation of plasma glucose) in both groups. However, only approximately 50% of glycogen was formed via the direct pathway in white muscle of exercised and nonexercised rats. Therefore prior exercise did not alter the pathways of tissue glycogen synthesis. To further study the incorporation of gluconeogenic precursors into muscle glycogen, exercised rats were infused with either saline, lactate (100 mumol.min-1.kg-1), or glucose (200 mumol.min-1.kg-1), containing [6-3H]glucose and [14C(U)]lactate. Plasma glucose was elevated one- to twofold and three- to fourfold by lactate and glucose infusion, respectively. Plasma lactate levels were elevated by about threefold during both glucose and lactate infusion. Glycogen was partially synthesized via an indirect pathway in white muscle and liver of glucose- or lactate-infused rats but not in saline-infused animals. Thus participation of an indirect pathway in white skeletal muscle glycogen synthesis required prolonged elevation of plasma lactate levels produced by nutritive support.