A new assay method for the measurement of thymidine kinase (TK) is described. Cytosols were prepared from TK- and TK+ cells and evaluated for TK activity using an assay which is based on the phosphorylation of [125I]-iododeoxyuridine, [125I]-iododeoxycytidine, or [3H]thymidine and the precipitation of the monophosphates of these nucleosides by lanthanum chloride. The specificity, reproducibility, sensitivity, and convenience of this assay are demonstrated.