Binding of [3H] folate to proteins in umbilical cord serum was studied in equilibrium dialysis experiments (37 degrees C, pH 7.4). A Scatchard analysis revealed the presence of high affinity (Kass = 9.10(10)1/mol, N = 0.5 nmol per 1 folate) and low affinity binding sites. High affinity binding was almost completely inhibited in the presence of methotrexate. A high affinity folate binding protein (saturated at a folate concentration of 10(-11)mol/l) appeared in the front effluent after application of serum to a DEAE-Sepharose Cl-6B column equilibrated with 0.05 mol/l imidazole buffer (pH, 6.3, 30 mmol/l NaCl), whereas low affinity binding activity mainly associated with albumin eluted from the column following a rise in the NaCl concentration to 1 mol/l. Low affinity binding predominated at folate concentrations above (10(-9) mol/l. The apparent molecular size of the high affinity binder was 35,000 as determined by gel filtration. The physiological role of the high affinity binder is obscure. However, it may represent an intracellular protein regulating folate stores, and its appearance in serum may be incidental to tissue turnover.