This chapter discusses the use of radioactive folate and binding proteins for the determination of folate. The concentration of folate in the serum and red cells is an important clinical tool in the diagnosis of megaloblastic anemia and for the evaluation of nutrition. This chapter defines the folate radioassay methodology, and discusses the advantages and disadvantages of the modifications introduced related to this methodology. The folate radioassay methodology is a proven and reliable procedure. It utilizes the commercially available high specific activity tritiated pteroylglutamic acid as the radioactive tracer, methyltetrahydrofolate as the standard for serum folate assays, and utilizes heat extraction only when there is an elevation of unsaturated serum folate-binding protein present. It is rapid and reproducible, and in an emergency a single sample can be assayed without the necessity for a new standard curve. In contrast to the microbiologic assay, it is not affected by antibiotics or turbidity and only minimally so by the antineoplastic drugs other than the folate antagonists. The use of gamma-labeled folates simplifies the radioassay methodology by eliminating liquid scintillation spectrometry. l25I-labeled folic acid, as either the monotyramide or the histamine conjugate, and 75Se-labeled folate, as the nonphysiologic pteroyl-L-methylselenocysteine, are now available.