The administration in vivo of either adrenaline or glucagon alone resulted in increases of about 2-fold in the amounts of active, non-phosphorylated, pyruvate dehydrogenase in the livers of fed male or female rats, whereas when administered together increases of about 4-fold were obtained. Ca2+-dependent increases in the amount of active enzyme of up to about 5-fold could be achieved in isolated rat liver mitochondria by incubating them with increasing extramitochondrial [Ca2+]; from this, two conditions of Ca loading were chosen which caused increases in active enzyme similar to those with the hormone treatments given above. The increases in enzyme activity owing to these Ca loads persisted through the 're-isolation' of mitochondria and their incubation in Na+-free KCl-based media containing EGTA. Differences from values obtained with unloaded controls could be diminished by adding Na+ ions to cause the egress of Ca2+ from the mitochondria, or enough extramitochondrial Ca2+ to saturate the enzyme in its Ca2+-dependent activation; the effects of Na+ could be blocked by diltiazem, an inhibitor of mitochondrial Na+/Ca2+ exchange. The re-isolated, Ca-preloaded, mitochondria also exhibited enhanced activities of 2-oxoglutarate dehydrogenase when assayed at non-saturating [2-oxoglutarate] by two different methods; effects of Na+, Ca2+ or diltiazem on the persistent activations of this enzyme were similar to those for pyruvate dehydrogenase. Na+ caused a marked depletion, which could be blocked by diltiazem, of the 45Ca content of re-isolated mitochondria which had pre-loaded with Ca, containing 45Ca, to the same degrees as above. The activities of pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase in incubated liver mitochondria prepared from rats subjected to the hormone treatments given above were found to behave in a very similar manner to those exhibited in the re-isolated, Ca-preloaded, mitochondria. It is concluded that these hormones each bring about the activations of these rat liver enzymes by causing increases in intramitochondrial [Ca2+], and that their effects, as such, are additive.