DNA-affinity chromatography has been used for the purification of DNA-binding proteins that control various cellular processes. There have been improvements in coupling methods and choice of supports over the years. The procedure for coupling 5'-aminoethyl-(dT)18 to silica activated with N-hydroxysuccinimide and a carbodiimide has been described. Also, the cyanogen bromide mediated coupling of aminoethyl-(dT)18 to Sepharose is described. Determination of (dT)18-coupling to silica and Sepharose is by 5' end-labeling an oligonucleotide containing a (dA)18 stretch of sequence and determining how much hybridizes with the (dT)18 support. Enzymatic synthesis of a double-stranded DNA-silica or Sepharose prevents modification of nucleotide bases. We have explained the use of DNA and RNA templates for template-directed enzymatic synthesis of affinity columns. DNA-affinity chromatography is a powerful method with broad applicability and we are currently extending this technology for purifying transcription factors, polymerases, and nucleases.