Conversion of lactate to glucose was examined in myotubes, minced muscle tissue, and rats exposed to ²H₂O or ¹³C-enriched substrates. Myotubes or minced skeletal muscle incubated with [U-¹³C₃]lactate released small amounts of [1,2,3-¹³C₃] or [4,5,6-¹³C₃]glucose. This labeling pattern is consistent with direct transfer from lactate to glucose without randomization in the TCA cycle. After exposure of incubated muscle to ²H₂O, [U-¹³C₃]lactate, glucose and glutamine, there was minimal release of synthesized glucose to the medium based on a low level of ²H enrichment in medium glucose but 50- to 100-fold greater ²H enrichment in glucosyl units from glycogen. The ¹³C enrichment pattern in glycogen from incubated skeletal muscle was consistent only with direct transfer of lactate to glucose without exchange in TCA cycle intermediates. ¹³C NMR spectra of glutamate from the same tissue showed flux from lactate through pyruvate dehydrogenase but not flux through pyruvate carboxylase into the TCA cycle. Carbon from an alternative substrate for glucose production that requires metabolism through the citric acid cycle, propionate, did not enter glycogen, suggesting that TCA cycle intermediates do not exchange with phosphoenolpyruvate. In vivo, the ¹³C labeling patterns in hepatic glycogen and plasma glucose after administration of [U-¹³C₃]lactate did not differ significantly. However, skeletal muscle glycogen was substantially enriched in [1,2,3-¹³C₃] and [4,5,6-¹³C₃]glucose units that could only occur through skeletal muscle glyconeogenesis rather than glycogenesis. Lactate serves as a substrate for glyconeogenesis in vivo without exchange into symmetric intermediates of the TCA cycle.