Abstract: Production of NADPH in Saccharomyces cerevisiae cells grown on glucose has been attributed to glucose-6-phosphate dehydrogenase (Zwf1p) and a cytosolic aldehyde dehydrogenase (Ald6p) [Grabowska and Chelstowska, 2003, J. Biol. Chem. 278, 13984-13988]. This was based on compensation by over-expression of Ald6p for phenotypes associated with ZWF1 gene disruption and on the apparent lethality resulting from co-disruption of ZWF1 and ALD6 genes. However, we have found that a zwf1ald6 mutant can be constructed by mating when tetrads are dissected on plates with a nonfermentable carbon source (lactate), a condition associated with expression of another enzymatic source of NADPH, cytosolic NADP+-specific isocitrate dehydrogenase (Idp2p). We previously demonstrated that a zwf1idp2 mutant loses viability when shifted to medium with oleate or acetate as the carbon source apparently due to the inadequate supply of NADPH for cellular antioxidant systems. In contrast, the zwf1ald6 mutant grows as well as the parental strain in similar shifts. In addition, the zwf1ald6 mutant grows slowly but does not lose viability when shifted to culture medium with glucose as the carbon source, and the mutant resumes growth when the glucose is exhausted from the medium. Measurements of NADP(H) levels revealed that NADPH may not be rapidly utilized in the zwf1ald6 mutant in glucose medium, perhaps due to a reduction in fatty acid synthesis associated with loss of Ald6p. In contrast, levels of NADP+ rise dramatically in the zwf1idp2 mutant in acetate medium, suggesting a decrease in production of NADPH reducing equivalents needed both for biosynthesis and for antioxidant functions.