The expression of cytosolic human thymidine kinase (TK) occurs in a cell cycle-dependent manner. Here, we show that TK is hyperphosphorylated during the M phase in several human cell lines. Our data from characterizing TK phosphorylation in proliferating and M phase-arrested HeLa cells suggest that the polypeptide of TK is differentially phosphorylated during the progression of the cell cycle. TK in the M phase-arrested HeLa cells was found to have a 10-fold lower affinity for its substrate, thymidine, than in the proliferating cells. We propose that phosphorylation of TK by the mitotic kinase(s) may provide an attenuating mechanism to prevent unnecessary synthesis of dTTP at the time of mitosis.