Abstract The interactions of glutamate metabolism with transport steps across the mitochondrial membrane and enzyme steps of the citric acid cycle have been investigated in rat heart mitochondria in order to elucidate factors regulating transfer of reducing equivalents into mitochondria via the malateaspartate cycle. With glutamate as substrate, net efflux of α-ketoglutarate from the mitochondria in State 3 was stimulated by addition of extramitochondrial malate, the concentration for a half-maximal effect being 0.36 ± 0.07 mm. Stimulation of α-ketoglutarate-malate exchange by a decrease of the malate gradient resulted in a fall of the intramitochondrial α-ketoglutarate concentration and diminished flux through α-ketoglutarate dehydrogenase. A value of 0.67 mm for the apparent Km of α-ketoglutarate dehydrogenase for α-ketoglutarate was calculated from these data. Glutamate transamination was inhibited in the uncoupled state and the intramitochondrial aspartate content increased up to 20-fold. The increased aspartate gradient across the mitochondrial membrane observed in the energy-depleted state was abolished, while aspartate efflux and glutamate transamination were restored either by direct addition of ATP or by addition of acetylcarnitine to increase substrate level phosphorylation. These effects were prevented by oligomycin, indicating that activation of aspartate efflux required energy in a form closely associated with the energy transfer reactions of oxidative phosphorylation. Regulation of flux through citrate synthase, aspartate aminotransferase, and α-ketoglutarate dehydrogenase as a function of the state of phosphorylation of the mitochondrial adenine nucleotides was investigated at high NADH:NAD ratios produced by State 4 and oligomycin inhibition, and at low NADH:NAD ratios produced by addition of uncoupling agents in the presence and absence of oligomycin. Acetylcarnitine and acetoacetate were used to provide sources of intramitochondrial acetyl coenzyme A. The following conclusions may be drawn from the data. In addition to the malate concentration gradient, net α-ketoglutarate efflux is regulated by the rate of generation of intramitochondrial α-ketoglutarate relative to the activity of α-ketoglutarate dehydrogenase as determined by product inhibition. Flux through α-ketoglutarate dehydrogenase was inhibited at high ATP:ADP ratios due to the high succinyl-CoA to CoA ratio, with the inhibition potentiated by an increase of the NADH:NAD ratio and relieved by an increased availability of intramitochondrial α-ketoglutarate. Production of α-ketoglutarate via citrate synthase was also regulated by the phosphorylation state of the intramitochondrial adenine nucleotides as indicated by succinyl-CoA inhibition rather than by direct ATP inhibition. Since inhibition of citrate synthase by succinyl-CoA has been shown to be competitive with acetyl-CoA, it is proposed that regulation is exerted by changes of the succinyl-CoA to acetyl-CoA ratio. This regulation is complementary to control of oxalacetate availability by the intramitochondrial NADH:NAD ratio which also regulates α-ketoglutarate production via both citrate synthase and aspartate aminotransferase. Coordination of flux between enzymes which produce intramitochondrial α-ketoglutarate, namely citrate synthase and aspartate aminotransferase, and α-ketoglutarate dehydrogenase which utilizes it, is determined by complex feedback relationships regulated indirectly both by the ATP:ADP ratio and the NADH:NAD ratio. These provide great flexibility to regulation of the over-all malate-aspartate cycle by controlling efflux of α-ketoglutarate from the mitochondria.