Saccharomyces cerevisiae possesses two cytosolic 5,10-methylenetetrahydrofolate (CH2-THF) dehydrogenases that differ in their redox cofactor specificity: an NAD-dependent dehydrogenase encoded by the MTD1 gene and an NADP-dependent activity as part of the trifunctional C1-THF synthase encoded by the ADE3 gene. The experiments described here were designed to define the metabolic roles of the NAD- and NADP-dependent CH2-THF dehydrogenases in one-carbon interconversions and de novo purine biosynthesis. Growth studies showed that the NAD-dependent CH2-THF dehydrogenase is interchangeable with the NADP-dependent CH2-THF dehydrogenase when flow of one-carbon units is in the oxidative direction but that it does not participate significantly when flux is in the reductive direction. 13C NMR experiments with [2-13C]glycine and unlabeled formate confirmed the latter conclusion. Direct measurements of cellular folate coenzyme levels revealed substantial levels of 10-formyl-THF (CHO-THF), the one-carbon donor used in purine synthesis, in the purine-requiring ade3 deletion strain. Thus, CHO-THF is necessary but not sufficient for de novo purine synthesis in yeast. Disruption of the MTD1 gene in this strain resulted in undetectable CHO-THF, indicating that the NAD-dependent CH2-THF dehydrogenase was responsible for CHO-THF production in the ade3 deletion strain. Finally, we examined the ability of wild-type and catalytically-inactive domains of the cytoplasmic C1-THF synthase to complement the adenine auxotrophy of the ade3 deletion strain. Both the dehydrogenase/cyclohydrolase (D/C) domain and the synthetase domain could functionally replace the full-length protein, but, at least for the D/C domain, complementation was not dependent on catalytic activity. These results reveal a catalytic role for the NAD-dependent CH2-THF dehydrogenase in the oxidation of cytoplasmic one-carbon units and indicate that the cytoplasmic C1-THF synthase plays both catalytic and noncatalytic roles in de novo purine biosynthesis in yeast.