The mechanism of posttranscriptional regulation of thymidine kinase (TK) enzyme levels following serum stimulation of quiescent cells has been investigated using stably transfected Rat 3 (TK-) cells containing the human TK complementary DNA linked to a hybrid SV40/human TK promoter. These cells expressed a wild-type human TK mRNA at relatively constant levels during the first G1 and S phase after serum stimulation. In contrast, TK enzyme activity and protein levels were low during G1 and increased dramatically as the cells entered S. A comparison of the patterns of protein expression (by Western blot) and enzyme activity indicated that the specific activity of the protein did not vary between G1 and S. A combination of pulse labeling and pulse-chase experiments indicated that the increase in TK protein levels at the G1-S transition was primarily the result of a stabilization of the protein at that time. The stability of a mutant form of TK lacking 16 NH2-terminal amino acids was regulated similarly to the wild-type, indicating that this region of the protein is not required for the regulation of protein turnover. Finally, indirect immunofluorescent labeling demonstrated that TK is uniformly distributed in the cytoplasm during both G1 and S phase.