1. Rat liver mitochondria oxidizing malate produce PEP (phosphoenolpyruvate) without the addition of ATP or other nucleotides. 2. The addition of oligomycin in the presence of 2,4-dinitrophenol did not abolish PEP formation and in some instances stimulated its formation. 3. Formation of PEP was inhibited by arsenate. 4. Arsenite decreased PEP formation and caused accumulation of pyruvate. 5. Added GTP and ITP had no effect on PEP formation. 6. PEP formed from malate in the presence of GTP and labelled Pi had a specific radioactivity approximately the same as the Pi with no contribution from the phosphate of the added GTP. 7. There was no parallelism between the effects of inhibitors on PEP formation from malate and their effects on the assayed activity of PEP carboxykinase. 8. In a direct comparison it was shown that the PEP carboxykinase content of mitochondria was insufficient to account for the PEP formation from malate. 9. Consideration of the kinetic characteristics of PEP carboxykinase and mitochondrial content of oxaloacetate and GTP show that this enzyme cannot account for the PEP formed from malate by mitochondria.

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