Dihydrofolate reductase from bovine liver has been purified 5000-fold employing conventional techniques and methotrexate/aminohexyl/Sepharose affinity chromatography. Electrophoresis of the isolated enzyme on polyacrylamide gels resulted in the separation of two enzymatically active protein components which were not interconvertible by treatment with dihydrofolate and/or the coenzyme. The two forms, present in a ratio of 20:1, were found by isoelectric focusing to have isoelectric points of 7.15 and 5.94. They had identical specific activities toward dihydrofolate (26.1-27.0 U/mg) and folate (1.3-2.2 U/mg), and had identical molecular weights (23500) and amino acid compositions. Due to the small quantity of the acidic form and the similarity of the two forms, the amino-terminal sequence (19 residues) was determined on a mixture of carboxymethylated reductase. The single sulfhydryl group of the enzyme can be modified by several sulfhydryl reagents in the native enzyme without loss of activity. Modification of the same residue occurs in the denaturated state and partially inhibits renaturation to the fully acitve enzyme. One disulfide bridge was detected by reduction and alkylation. The cleavage of this bond did not effect the enzymatic activity.