A radioassay for the measurement of serum folate levels using commercially available beta lactoglobulin, as the folate-binding protein, and 3H-pteroylglutamic acid (3H-PGA) is reported. The assay was run in a one-step simultaneous addition at room temperature. A standard curve was constructed to a sensitivity of 0.25-10 ng of N-methyltetrahydrofolic acid (methyl-THFA). There was a clear correlation with separation into normal (greater than 6 ng/ml), indeterminate range (3-6 ng/ml), and deficient (0-3 ng/ml), as measured by radioassay. Serums from patients receiving antibiotics had normal folate levels with this assay. 3H-PGA was also used to measure serum folic acid-binding protein (FABP). In normal serums, the mean FABP was 18 pg bound/0.4 ml serum, while in folate-deficient serums it was 133 pg bound/0.4 ml of serum. Folate-deficient patients had a fall in serum FABP to the normal range when treated with folic acid. Serum FABP, in a patient on a folate-deficient diet, increased with early folate deficiency and abruptly fell to normal with a regular diet. FABP in lysates of folate-deficient bone marrow was higher than normal marrow. Patients with B12 deficiency, multiple myeloma, cirrhosis, pregnancy, or taking oral contraceptives had normal FABP. Elevated FABP was found in two out of ten patients taking Dilantin. This radioassay and the measurement of FABP should simplify the diagnosis of folate deficiency.