The methenyltetrahydrofolate cyclohydrolase activity of the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase domain catalyzes the conversion of 5,10-methenyltetrahydrofolate to 10-formyltetrahydrofolate. We have observed that in the presence of this domain, 10-formyltetrahydrofolate is converted to 5-formyltetrahydrofolate. The effects of nucleotide analogs on the initial rates of this reaction were found to be similar to their effects on the cyclohydrolase activity, establishing that 5-formyltetrahydrofolate production is dependent on the cyclohydrolase. The specific activity of 5-formyltetrahydrofolate production is approximately 7 × 10 4 -fold lower than that of the cyclohydrolase activity but can be used to obtain quantitative conversion of 10-formylH 4 folate to 5-formyltetrahydrofolate, in mg amounts. This “side-reaction” may contribute to thein vivoproduction of (6S)-5-formyltetrahydrofolate.