Multienzyme complex responsible for DNA biosynthesis was isolated from the nuclei of S phase LP1-1 cells, in which LMTK^- cells was transfected with Herpes simplex viral thymidine kinase gene (HSVTK). This complex had a number of enzyme activities i.e., DNA polymerase α, protein kinase, thymidine kinase and topoisomerase II, and could catalyse the incorporation of labelled precursors into DNA. This fraction, defined as "replitase", contained spherical particles of ranging from 33.7 to 50.6 ㎚ in diameter, apparently multienzyme complex for de novo DNA biosynthesis. 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. Here we presented the evidence for the functional association of one of the enzyme activities, DNA polymerase α and thymidine kinase, with several other enzyme activities at replitase and nuclear matrix levels. Hydroxyurea, novobiocin and aphidicolin, inhibitors of ribonucleotide reductase, topoisomerase and DNA polymerase α respectively, all inhibited DNA polymerase α in nuclear matrix and in replitase. In contrast to these inhibition, topoisomerase II was not affected by aphidicolin and hydroxyurea in nuclear matrix. On the allosteric interaction of thymidine kinase, hydroxyurea inhibited thymidine kinase in replitase, but not in nuclear matrix. However, novobiocin inhibited thymidine kinase in nuclear matrix, but not in replitase. These results indicate the allosteric interaction between the subunits of a multienzyme DNA-synthesizing complex, which could be modulated by the specific inhibitors of individual enzyme activities in intact cells.