A multienzyme complex responsible for DNA replication was isolated from the nuclei of synchronized LP1-1 cells, in which LMTK^- cell was transfected with Herpes simplex viral thymidine kinase gene (HSVTK). The nuclear fraction was found to contain spherical particles of ranging from 33.7 to 50.6 ㎚ in diameter, apparently multienzyme complex for de novo DNA biosynthesis. This multienzyme complex, termed as `replitase`, had several enzyme activities of DNA polymerase α, protein kinase, thymidine kinase and topoisomerase II, and thus, could catalyse the incorporation of labelled precursors into DNA. DNA polymerase α and its associated enzyme activities were unique to cells in S phase and increased coordinately during the G1/S phase transition of the cell cycle, suggesting the functional association of thymidine kinase with DNA polymerase α at the levels of multienzyme complex and nuclear matrix. Hydroxyurea, aphidicolin and novobiocin, inhibitors of ribonucleotide reductase, DNA polymerase α and δ, and topoisomerase, respectively, all inhibited thymidine kinase and DNA polymerase α activities in intact S phase LP1-1 cells. However, in permeabilized cells hydroxyurea had no inhibitory effect on the activity of either enzyme, while aphidicolin, novobiocin and TK antiserum inhibited the activities of thymidine kinase and DNA polymerase α. The similar inhibitions observed in permeabilized cells were seen in the preparations of multienzyme complex and nuclear matrix. These results support the hypothesis that transfected viral thymidine kinase acts as a component of the multienzyme complex responsible for DNA replication and has an allosteric interaction with the subunits of the multienzyme complex.