The adsorption of thymidylate synthetase from Escherichia coli B to aminoalkyl-Sepharose with the increasing length of carbon chain (2--6 carbon atoms) was investigated. A correlation was found between the chain length and adsorption effectiveness, increasing from the two- to the six-carbon chain. A hydrophobic chromatography of the enzyme on aminobutyl-Sepharose gave about 20-fold purification. A new affinity chromatography carrier was synthesized containing tetrahydromethotrexate linked to aminoethyl-Sepharose via its carboxylic groups. The carrier adsorbed the enzyme from the crude preparation only in the presence of deoxyuridine 5'-monophosphate (dUMP) in a concentration of 2 X 10(-5) M. The specifically adsorbed thymidylate synthetase was eluted with sacharose-containing buffers in which dUMP was omitted. The purification procedure was applied to a crude thymidylate synthetase preparation from resting E. coli, calf thymus, Sarcoma 180, and Gardner lymphosarcoma. The purified enzyme from all mentioned sources showed one protein band on disc electrophoresis corresponding to enzymatic activity. The formation of a reversible noncovalent complex enzyme-tetrahydromethotrexate-dUMP on the affinity column is supposed.