Thymidine kinase was purified to near homogeneity by affinity chromatography from cytosol fraction of rat hepatoma 3924A. The enzyme had a Mr of 81,000 and was composed of two subunits of Mr = 44,000. Antiserum made against it neutralized the activities of thymidine kinase from both rat livers and hepatomas. Neutralization studies with the antiserum revealed that hepatic transformation resulted in 4-, 15- and 25-fold increase in the amount of cytosol thymidine kinase in hepatomas 16, 7787 and 3924A of slow, medium and fast growth rate, respectively.