The protein-dependent retention of double-stranded DNA molecules on nitrocellulose filters has been used to show that pure dihydrofolate reductase from Lactobacillus casei has affinity for DNA. Dihydrofolate reductase will bind to end-labeled linear double-stranded DNA and to DNA in supercoiled form. Coenzymes and certain inhibitors do not affect the affinity of the protein to DNA, indicating that the DNA-binding region of the protein is distinct from the binding sites for these molecules. Comparison of the retention on filters by dihydrofolate reductase of two plasmid DNAs, differing only in a 3000-base pair insert containing the L. casei gene for dihydrofolate reductase, showed that in the presence of this DNA region lower concentrations of the protein were required to give significant retention; it is possible that a specific DNA-protein interaction underlies this effect. This presents the possibility of studying the interaction with DNA of a protein for which a crystal structure and considerable nuclear magnetic resonance data are already available.