During gluconeogenesis from L-glutamine, 14CO2 is fixed into glucose. Inhibitors of pyruvate transport or pyruvate carboxylase only slightly decrease the 14CO2 incorporation, indicating that a pathway of formation of pyruvate, followed by pyruvate carboxylation, is not primarily involved. These results suggest that 14CO2 fixation is effected by a reverse (exchange) reaction of P-enolpyruvate carboxykinase. MnCl2 (0.5 mM) stimulates the 14CO2 fixation in glucose from L-glutamine by nearly 50%. This result is in accord with a recent study (Colombo, G., Carlson, G. M., and Lardy, H. A. (1981) Biochemistry 20, 2749-2757) showing that Mn2+ greatly stimulates the reverse reaction (P-enolpyruvate leads to oxalacetate) of purified rat liver P-enolpyruvate carboxykinase. Preliminary calculations suggest that 14CO2 is also fixed by reversible P-enolpyruvate carboxykinase activity during gluconeogenesis from L-lactate, in addition to the fixation of H14CO3(-) in the pyruvate carboxylase forward reaction.