1. Dihydrofolate reductase was purified from Lactobacillus casei MTX/R, and studied on affinity columns containing folic acid and methotrexate. Two forms of the enzyme were interconverted by incubation with substrates. 2. Affinity columns were prepared from agarose activated with cyanogen bromide and coupled with 1,6-diaminohexane. Stable folate derivatives were covalently attached by using a carbodi-imide condensation. 3. Columns containing folic acid retarded but did not retain the enzyme. 4. Methotrexate at pH 6.0 was particularly effective for retention of the enzyme. 5. There is selective loss of one form of the enzyme during affinity chromatography in the absence of added NADPH. This loss is due to conversion into a single enzyme form on the column. 6. NADPH has a dual effect in stabilizing the enzyme and in sensitizing it to inactivation by methotrexate, particularly in the presence of glycine. 7. Protein with affinity for methotrexate, but without dihydrofolate reductase activity, may also be eluted from the columns. 8. In a single-step procedure the enzyme was purified nearly 4000-fold from mammalian skin.