This chapter focuses on radioenzymatic assay for dihydrofolate reductase using [3H]dihydrofolate. Folic acid (pteroylglutamic acid, PteGlu) or dihydrofolic acid (H2PteGlu) are reduced to tetrahydrofolic acid (H4PteGlu) by reduced nicotinamide adenine dinucleotide phosphate (NADPH) with the enzyme dihydrofolate reductase (DHFR) catalyzing the reaction. The product, H4PteGlu, can be separated from the substrate by precipitation of the PteGlu or H2PteGlu with zinc sulfate. This method permits monitoring the reaction by the direct measurement of the reduction of tritium-labeled PteGlu or H2PteGlu. The enzymatic reduction of the H2PteGlu is then initiated by the addition of NADPH and DHFR. Unlike the enzymatic reduction of PteGlu, the reduction of H2PteGlu by DHFR is optimal at neutral pH. There are several advantages of this radioenzymatic assay: (1) measurement of the activity of the enzyme using H2PteGlu, the more physiological substrate, (2) the coupling of the chemical reduction of [3H]PteGlu to [3H]H2PteGlu with the enzymatic reaction eliminates the need for the complicated synthesis and storage of the labile [3H]H2PteGlu, (3) depending on the specific activity of [3H]PteGlu, the assay is very sensitive and can monitor the reduction of as little as 0.5 nM of substrate, and (4) direct measurement of the reduction of the substrate rather than the oxidation of NADPH, so that enzyme activity can be determined in crude tissue or cell preparations, which may contain other components which can oxidize NADPH.