Mitochondria of fungi contain two respiratory chain enzymes concerned with the oxidation of matrix NADH. These are the proton-pumping NADH:ubiquinone oxidoreductase, also called complex I, which has a high affinity for NADH, and a non-proton-pumping NADH:ubiquinone oxidoreductase, called alternative NADH dehydrogenase, which has a low affinity for NADH. The role of these two enzymes in normal and overflow catabolism has been studied in Aspergillus niger. Three strains were investigated, the wild-type 732, the mutant nuo51 that was generated from the wild-type by disrupting the gene of the (51-kDa) NADH-binding subunit of complex I and the citric acid over-producing strain B60 that looses complex I concomitantly with the onset of the over-production. Under standard growth conditions, respiratory energy transduction in the mutant nuo51 was decreased by 40% compared to the parental wild-type and the strain B60. Respiratory electron transfer in the mutant nuo51, however, meets standard catabolic requirements. The intracellular levels of citric acid cycle intermediates in the mutant nuo51 were the same as in the other two strains. Under growth conditions which lead to uncontrolled catabolic flux through glycolysis, a dramatic catabolic overflow occurred in the mutant nuo51. Intracellular levels of citric acid cycle intermediates increased to 20-fold normal levels. The strain B60, likewise lacking complex I under these conditions, excretes large amounts of citrate to moderate the intracellular catabolic overflow.