Activity of glycine decarboxylase decreased by 60-70% after the isolated pea leaf mitochondria were aged for 5 h in the absence of glycine and was completely lost after 24 h. The reverse reaction, i.e., production of glycine from serine, ammonium, dihydrolipoate, and bicarbonate, was also inhibited in these aged mitochondria. Glycine decarboxylase could be reactivated by both exogenous and endogenous ATP. The latter was formed during the oxidation of succinate, malate, or oxoglutarate. Glycine decarboxylase consists of four subunits (P-, H-, L-, and T-proteins). The aged mitochondria were able to catalyze the exchange of [14C]-bicarbonate-glycine and the oxidation of dihydrolipoate, indicating the persistence of P-, H-, and L-protein activities. Serine hydroxymethyltransferase catalyzes the formation of serine from methylene tetrahydrofolate and another glycine and molecule at the last reaction of glycine oxidation. The aged mitochondria were able to catalyze the formation of methylene tetrahydrofolate from [14C]serine and its reverse reaction. Therefore, it was concluded that the loss of glycine decarboxylase activity was due to an inhibition of the reaction catalyzed by T-protein, which required ATP for its activation.