The removal of the 1-carbon of threonine can occur via threonine dehydrogenase or threonine aldolase, this carbon ending up in glycine to be liberated by the mitochondrial glycine cleavage system and producing CO₂. Alternatively, in the threonine dehydratase pathway, the 1-carbon ends up in -ketobutyrate, which is oxidized in the mitochondria to CO₂. Rat hepatocytes, incubated in Krebs-Henseleit medium, were incubated with 0.5 mM L-[1-¹⁴C]threonine, and ¹⁴CO₂ production was measured. Added glycine (0.3 mM) marginally suppressed threonine oxidation. Cysteamine (0.5 mM), a potent inhibitor of the glycine cleavage system, reduced threonine oxidation to 65% of controls. However, -cyanocinnamate (0.5 mM), a competitive inhibitor of mitochondrial -keto acid uptake, reduced threonine oxidation to 35% of controls. These data provided strong evidence that ~65% of threonine oxidation occurs through the glycine-independent threonine dehydratase pathway. Glucagon (10⁷ M) increased threonine oxidation and stimulated threonine uptake by these cells. In summary, the majority of threonine oxidation occurs through the threonine dehydratase pathway in rat hepatocytes, and threonine oxidation is increased by glucagon, which also increases threonine's transport.