To define the functional aspect of transfected viral thymidine kinase, we have prepared a DNA-synthesizing multienzyme complex from the nuclei of synchronized LP1-1 cells. This complex was previously shown to contain several enzyme activities, including DNA polymerase α protein kinase, topoisomerase and thymidine kinase in our laboratory. The activity of thymidine kinase was parallel to that of DNA polymerase α during the cell cycle, when the whole nuclear fraction or the nuclear matrix was used for examination. By immunoblot analysis with a specific antiserum raised against the purified tribrid fusion protein (recA-TK-β·gal), viral thymidine kinase was identified as a 43Kd polypeptide vi the fraction of the DNA polymerase-associated multienzyme complex prepared from LP1-1 cells, but not from LMTK^- cells. These results provide evidence for the expression of the transfected viral thymidine kinase gene in the association with the host DNA-synthesizing multienzyme complex.