The lysates of peripheral cells as well as the serum from some patients with chronic myelogenous leukemia, contained a macromolecular factor which bound tritiated folic acid. Bound tracer folate filtered through Sephadex G-75 and G-100 columns with the early effluent and appeared with the inner volume through a Sephadex G-200 column. Bound tracer could not be extracted from solution by coated charcoal or the anion exchange resin Dowex 2-X8 and could not be reduced to tetrahydrofolate by folate reductase. The velocity of the binding reaction was very rapid and dissociation of bound tracer extremely slow. Binding decreased sharply below pH 5.0 and the binding factor as well as the folate-binder complex, resisted 56 degrees C for 30 min. The binding factor in the leukemic lysate could be separated from endogenous folate reductase by filtration through a G-75 Sephadex column. Competitive inhibition studies demonstrated little or no inhibition of binding of tritiated folic acid by formyltetrahydrofolate and methyltetrahydrofolate. Diopterin (pteroyldiglutamate), pteropterin (pteroyltriglutamate), methotrexate, and dihydrofolate inhibited binding of tracer folate but not as effectively as unlabeled folic acid. The function of this folate binder is unknown. However, that it reacts with dihydrofolate suggests some relationship (physiologic or pathologic) to DNA synthesis since this folate cofactor is essential for the de novo synthesis of thymidylate from deoxyuridylate. In addition, these findings also suggest that the binding of methotrexate may, like folate, inhibit its reaction with folate reductase, and thus be a mechanism by which leukemic cells become resistant to this drug.