Thymidine kinase from a transplantable colon adenocarcinoma, induced by 1,2-dimethylhydrazine and maintained in CDF rats, was purified by affinity chromatography using thymidine-3'(4-aminophenylphosphate) coupled to carboxyhexyl-Sepharose. Most of the contaminating protein passed through the column; non-specifically adsorbed protein was washed from the column by 0.1 M KC1 in 0.01 M Tris-HC1, 7.5. Thymidine kinase was eluted with 0.1 mM thymidine, 0.1 M KC1 in 0.01 M Tris-HC1, pH 7.5. The purified enzyme accounted for about 26% of the applied activity; the specific activity of the purified material (peak fraction) was 3,500 moles TMP formed per mg protein per 10 min., a 1,800-fold purification of the applied extract. The preparation is free of nucleoside phosphotransferase, but contains other protein impurities. Purification was completed in less than 1 hour, making this a useful procedure for isolation of this unstable enzyme.