Null mutations in the Kluyveromyces lactis RAG2 gene mutant and in the Saccharomyces cerevisiae PGI1 gene eliminate the phosphoglucose isomerase enzyme activity. This blocks glycolysis at the same point in both organisms. However, there is a big difference between the two mutant phenotypes. The K. lactis rag2Δ mutant can grow on glucose as a sole carbon source, while the S. cerevisiae pgiΔ mutant cannot. In several reports this difference has been ascribed to the ability of K. lactis mitochondria to oxidize cytosolic NADPH, while S. cerevisiae mitochondria are incapable of oxidizing NADPH. However, in these reports no direct measurements were done on the NADPH-oxidizing ability of the K. lactis mitochondria or on the effect of a rag2Δ mutation on this activity. In this report mitochondria were isolated both from the wild-type Kluyveromyces lactis strain CBS2359 and from an isogenic rag2Δ mutant, which were grown in an aerobic, glucose-limited chemostat culture. The measurements done on these mitochondria indicated that the NADPH dehydrogenase activity was 5 fold induced in the mutant. Interestingly, also a mitochondrial alcohol dehydrogenase activity was induced in the mutant, which together with an NADPH-dependent acetaldehyde-reductase in the cytosol could form a redox shuttle over the mitochondrial membrane. Therefore, apart from confirming the participation of a mitochondrial NADPH dehydrogenase in the oxidation of cytosolic NADPH in a K. lactis rag2Δ mutant, an acetaldehyde-ethanol shuttle was identified as a possible alternative mitochondrial NADPH-oxidising system.