13C nuclear magnetic resonance spectroscopy was used to study the activity of malic enzyme in isolated brain mitochondria from rat in the presence of unlabelled malate and [U-13C]glutamate. ADP, inorganic phosphate, malate and [U-13C]glutamate were added to a suspension of oxygenated mitochondria. Typical tricarboxylic acid (TCA) cycle constituents (malate, 2-oxoglutarate and succinate) were labelled from [U-13C]glutamate and detected in the superfusion medium. The labelling patterns in the different atom positions of glutamate revealed entry of both unlabelled and labelled acetyl-CoA into the TCA cycle. Unlabelled acetyl-CoA was derived via pyruvate from exogenously applied malate by the action of mitochondrial malic enzyme, while labelled acetyl-CoA was derived from TCA cycle intermediates, most likely by the action of mitochondrial malic enzyme on malate produced from [U-13C]glutamate. The results demonstrate malic enzyme activity and pyruvate recycling in isolated rat brain mitochondria.