Two thymidine kinase isoenzymes, TK 3 and TK 4, from mononuclear leucocytes from a patient with acute monocytic leukemia, were purified and characterized in regard to the molecular weights and kinetic properties. The molecular weights of TK 3 and TK 4 were 60 000 and 45 000, respectively. In the presence of 2 mM ATP, the molecular weight of TK 3 increased to 200 000, whereas the molecular weight of TK 4 was unchanged. Studies of the kinetic properties showed clear differences between TK 3 and TK 4. With thymidine as substrate, TK 3 showed biphasic kinetics with a Km of 22 microM, and TK 4 showed Michaelis-Menten kinetics with a Km of 0.33 microM. With ATP as substrate, TK 3 showed Michaelis-Menten kinetics with a Km of 100 microM, and TK 4 showed biphasic kinetics with a Km of 3.5 microM. With dTTP as inhibitor, TK 3 showed cooperative inhibition kinetics, and TK 4 showed non-cooperative competitive inhibition kinetics. The dTTP concentration at 50% inhibition was 75 microM for TK 3 but 380 microM for TK 4. Comparison of the molecular weights and the kinetic properties of TK 3 and TK 4 with the corresponding data previously obtained for TK 1 and TK 2 from normal human lymphocytes indicate the existence of four thymidine kinase isoenzymes in human leucocytes.