Rapid and convenient purification procedures based upon heparin-agarose chromatography for C1-tetrahydrofolate synthase from Saccharomyces cerevisiae and 10-formyltetrahydrofolate synthetase from Clostridium acidi-urici have been developed. The purification of the yeast enzyme involves three chromatographic steps that can be done rapidly, with no intervening dialyses, and results in high yield. The first step alone, heparin-agarose chromatography, is sufficient to purify the enzyme from yeast bearing a cloned copy of the ADE3 gene that overexpresses the protein. The other steps in the purification from wild-type yeast are matrex gel red A and phenyl-Sepharose chromatography. The purification of the clostridial enzyme involves protamine sulfate fractionation and heparin-agarose chromatography. Heparin-agarose also binds two other enzymes that use tetrahydrofolate, 5,10-methenyltetrahydrofolate cyclohydrolase and 5,10-methylenetetrahydrofolate dehydrogenase. Thus, heparin-agarose should prove useful in purification of a variety of enzymes that utilize tetrahydrofolate or its derivatives as a cofactor.