It has been reported that the polypeptide of thymidine kinase type 1 (TK1) from human and mouse cells can be modified by phosphorylation. Our laboratory has further shown that the level of human TK phosphorylation increases during mitotic arrest in different cell types (Chang, Z.-F., Huang, D.-Y., and Hsue, N.-C. (1994) J. Biol. Chem. 269:21249-21254). In the present study, we demonstrated that a mutation converting Ser13 to Ala abolished the mitotic phosphorylation of native TK1 expressed in Ltk- cells. Furthermore, we expressed recombinant proteins of wild-type and mutated human TK1 with fused FLAG epitope in HeLa cells, and confirmed the occurrence of mitotic phosphorylation on Ser13 of hTK1. By using an in vitro phosphorylation assay, it was shown that wild-type hTK1, but not mutant TK1(Ala13), could serve as a good substrate for Cdc2 or Cdk2 kinase. Coexpression of p21(waf1/cip1), which is a universal inhibitor of Cdk kinases, in Ltk- fibroblasts also suppressed mitotic phosphorylation of hTK1 expressed in this cell line. Thus, Cdc2 or related kinase(s) is probably involved in mitotic phosphorylation on Ser13 of the hTK1 polypeptide. We also found that mutation on Ser13 did not affect the functional activity of hTK1. As the sequences around Ser13 are highly conserved in vertebrate TK1s, we speculate that phosphorylation of Ser13 may play a role in the regulation of TK1 expression in the cell cycle.