The liver cytosolic enzyme, 10-formyltetrahydrofolate dehydrogenase (10-FTHFDH) (EC 126.96.36.199) catalyzes two reactions: the NADP(+)-dependent oxidation of 10-formyltetrahydrofolate to tetrahydrofolate and CO2 and the NADP(+)-independent hydrolysis of 10-formyltetrahydrofolate to tetrahydrofolate and formate. It exists as a tetramer of 99-kDa subunits in rat liver. We expressed rat liver cDNA encoding 10-FTHFDH in insect cells using the pVL 1393 expression vector and MaxBac expression kit. Despite the absence of a leader peptide the recombinant 10-FTHFDH was released from the cells to the culture medium during production in both Sf9 and High five cell lines. The enzyme released into the medium was no less than 80% of the total recombinant 10-FTHFDH. Both enzyme pools, from the medium and from cell extracts, displayed high activity. The maximum expression of 10-FTHFDH was observed 72 h postinfection in High five cells and 96 h postinfection in Sf9 cells. High five cells revealed four times higher expression of the recombinant enzyme per milligram of total cell protein than Sf9 cells. Passage of the cell-free culture medium over an affinity column of 5-formyltetrahydrofolate-Sepharose provided 10-FTHFDH that was more than 95% pure. Additional purification on a Mono Q column resulted in an homogenous preparation of the enzyme. Purified recombinant 10-FTHFDH displayed both dehydrogenase and hydrolase activities, similar to those of the rat liver enzyme, and the recombinant enzyme remained active at least 12 months when stored appropriately. These results show that 10-FTHFDH can be overexpressed as a functional enzyme in baculovirus-infected insect cells and purified in two steps by simple chromatographic procedures.