Cells of the yeast Saccharomyces cerevisiae contain three NAD kinases; namely, cytosolic Utr1p, cytosolic Yef1p, and mitochondrial Pos5p. Previously, the NADH kinase reaction catalyzed by Pos5p, rather than the NAD kinase reaction followed by the NADP(+)-dependent dehydrogenase reaction, had been regarded as a critical source of mitochondrial NADPH, which plays vital roles in various mitochondrial functions. This study demonstrates that the mitochondrial NADH kinase reaction is dispensable as a source of mitochondrial NADPH and emphasizes the importance of the NAD kinase reaction, followed by the mitochondrial NADP(+)-dependent dehydrogenase reaction. Of the potential dehydrogenases (malic enzyme, Mae1p; isocitrate dehydrogenase, Idp1p; and acetaldehyde dehydrogenases, Ald4/5p), evidence is presented that acetaldehyde dehydrogenases, and in particular Ald4p, play a prominent role in generating mitochondrial NADPH in the absence of the NADH kinase reaction. The physiological significance of the mitochondrial NADH kinase reaction in the absence of Ald4p is also demonstrated. In addition, Pos5p is confirmed to have a considerably higher NADH kinase activity than NAD kinase activity. Taking these results together, it is proposed that there are two sources of mitochondrial NADPH in yeast: one is the mitochondrial Pos5p-NADH kinase reaction and the other is the mitochondrial Pos5p-NAD kinase reaction followed by the mitochondrial NADP(+)-dependent acetaldehyde dehydrogenase reaction.