Column chromatography utilizing polynucleotides immobilized on solid support is reviewed. This form of affinity chromatography is used for the isolation of polynucleotides and polynucleotide binding proteins, and to a lesser extent for analysis. Several specific applications within these categories have been widely used in the biomedical sciences. Poly(A) mRNA is routinely isolated using oligo(dT) or oligo(dU) supports. Many DNA binding proteins, including transcription factors, restriction endonucleases, and proteins involved in DNA repair, replication, recombination, and transposition have been purified using DNA affinity chromatography. Recently, DNA supports suitable for use in high-performance liquid chromatography have been described and utilized. The current usage of DNA affinity chromatography is reviewed and potential future uses for this technology are speculated upon.