C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."