The protein product of the ADE3 gene of the yeast Saccharomyces cerevisiae has been identified as the cytoplasmic trifunctional C1-tetrahydrofolate (THF) synthase, which possesses 10-formyl-THF synthetase (EC 6.3.4.3), 5,10-methenyl-THF cyclohydrolase (EC 3.5.4.9), and 5,10-methylene-THF dehydrogenase (EC 1.5.1.5) activities. However, it has been suggested that the ADE3-encoded C1-THF synthase does not play a role in providing the enzymes involved in the generation of one-carbon intermediates in the biosynthesis of the purine bases but functions in maintaining the structural integrity of the enzyme complex involved in purine biosynthesis [Barlowe, C. K. & Appling, D. A. (1990) Mol. Cell. Biol. 10, 5679-5687]. This hypothesis is based on their finding that the presence of the full-length ADE3 C1-THF synthase, whether catalytically active or not, is correlated with the Ade+ phenotype. In contrast to their results, our deletion analysis of the ADE3 gene indicates that the presence of either the synthetase or dehydrogenase/cyclohydrolase domains of C1-THF synthase is enough to complement the adenine requirement in ade3 strains. These results are also consistent with those obtained in heterologous expression of spinach and Clostridium acidiurici monofunctional synthetases in ade3 strains. Heterologous expression studies show that the high synthetase activity may be correlated with the increased growth in medium lacking adenine. These results suggest that the catalytic activity of the C1-THF synthase is involved in purine biosynthesis.