The path of glycogen synthesis from three-carbon precursors was studied via single-pass perfusions in three distinct rabbit skeletal muscle preparations, i.e., glycolytic (greater than 99% type IIb), oxidative (greater than 97% type I), and mixed (type I, IIa, and IIb). The extent of interaction between the Krebs cycle and glycogenesis was assessed utilizing [1-14C]- or [2-14C]lactate at basal (1.1 +/- 0.1 mM) and elevated (8.1 +/- 0.3 mM) lactate concentrations (protocols 1 and 2). Under conditions in which the net balance of glucose and lactate, [14C]lactate removal, and venous lactate-specific activity were similar, the yields of 14CO2 and [14C]glycogen were not significantly influenced by position of the label. Additional perfusions were performed with lactate (8.0 +/- 0.1 mM) and acetate (1.0 +/- 0.1 mM) as sole substrates and either [U-14C]lactate or [2-14C]acetate as the tracer. Under conditions of net glycogen synthesis, the incorporation of [14C]lactate into glycogen [in disintegrations/min (dpm).g-1.2 h-1] was 40,940 +/- 3,320, 1,540 +/- 320, and 32,600 +/- 4,100 in the glycolytic, oxidative, and mixed preparations, respectively. However, no incorporation of [2-14C]acetate into glycogen was observed in any preparation, despite a significant yield of 14CO2. Mercaptopicolinic acid, a potent inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), demonstrated no significant effect on net substrate balance, tracer uptake, net glycogen synthesis, incorporation of [14C]lactate and [3H]-glucose into glycogen, or 14CO2 yield. Current results suggest an extramitochondrial route for net glycogen synthesis from three-carbon precursors, exclusive of PEPCK, that is consistent across all mammalian skeletal muscle fiber types.