10-Formyltetrahydrofolate dehydrogenase (EC 126.96.36.199) is a bifunctional enzyme, displaying both NADP(+)-dependent dehydrogenase activity for the formation of tetrahydrofolate and CO2, and NADP(+)-independent hydrolase activity for the formation of tetrahydrofolate and formate. A previous report [Case, Kaisaki and Steele (1988) J. Biol. Chem. 263, 1024-1027] claimed that dehydrogenase and hydrolase activities were products of separate cytosolic and mitochondrial forms of this enzyme. Here we report that recombinant 10-formyltetrahydrofolate dehydrogenase carries out both enzymic reactions, proving that a product of a single gene, i.e. one protein, not two, has both activities. The stable synthetic analogue 10-formyl-5,8-dideazafolate can substitute for the labile natural substrate, 10-formyltetrahydrofolate, in both reactions. This was shown with both native and recombinant rat liver enzyme. The Km values for 10-formyl-5,8-dideazafolate were half of those for 10-formyltetrahydrofolate in both the dehydrogenase and hydrolytic reactions. The Vmax, values were similar for both substrates. Both dehydrogenase and hydrolase reactions were dependent on the presence of 2-mercaptoethanol. The pH optima were 7.8 and 5.6 for the dehydrogenase and hydrolase reactions respectively, consistent with the presence of two active sites in the enzyme.