AbstractDihydrofolate reductase, purified to homogeneity from amethopterin‐resistant Lactobacillus casei, was immobilized by coupling to cyanogen bromide‐activated Sepharose or carbodiimide‐activated CH‐Sepharose. Coupling yields were determined by amino acid analysis following the hydrolysis of the gel. Enzyme activity was measured by the conventional spectrophotometric procedure, thus permitting the facile characterization of the immobilized enzyme. The pH optimum of the immobilized enzyme was shifted to 5.8 compared with pH 5.5 for the soluble enzyme. The immobilized enzyme retained greater than 90%of the initial activity over a six‐month period and could be reused as many as ten times without loss of activity. As observed with the soluble enzyme, the activity of immobilized enzyme, which was lost on denaturation with 4M guanidine hydrochloride, was recovered rapidly and completely by washing the gel with buffer. The Kmapp values for dihydrofolate and NADPH for the immobilized enzyme were increased 15–164‐fold over the Km values measured for soluble dihydrofolate reductase. Scatchard analysis of the interaction of amethopterin with the immobilized enzyme yielded linear plots and a Kdapp value of 0.56 ×10−8M, and revealed that all of the immobilized enzyme molecules were capable of binding the ligand.