Abstract— Thymidine kinase (ATP: thymidine‐5′‐phosphotransferase EC 2.7.1.21) of the supernatant fraction from 6‐day‐old rat brain possessed a pH optimum of 8.0 and required the presence of 5mM‐ATP and 2.5 mM‐MgCl2 for maximum activity. The activity was completely inhibited by addition of 1.8 mM‐TTP. The enzyme activity was lost if the same supernatant fraction was refrozen and thawed. Km was 2.8 × 10−6 M for [6‐3H]thymidine. Following subcellular fractionation of rat brain, the greatest proportion and highest specific activity of thymidine kinase was found in the supernatant fraction. Thymidine kinase activities reached a maximum at 6 days of age and then dropped sharply during maturation. Comparative studies of thymidine kinase activities of cerebrum, cerebellum and the remainder of the brain during growth indicated that the activity in the cerebellum was usually higher than those in the cerebrum and the remainder, and the biggest differences obtained at 6 days after birth corresponded with the peak in cerebellar activity.