We have shown that MT depolymerization by colchicine and other drugs is sufficient to initiate DNA synthesis in serum-free cultures of embryonic fibroblasts and that stabilization of MTs with taxol inhibits this initiation. Growth factors and oncogenic DNA viruses also initiate DNA synthesis by a taxol-sensitive mechanism that appears to require MT depolymerization or rearrangements. Because we have shown that microtubule heterogeneity exists within single fibroblastic cells, we have carried out a series of experiments to determine the extent of microtubule disruption necessary to initiate DNA synthesis. We have compared the effects of various concentrations of colchicine and taxol on initiation of DNA synthesis with their effects on cytoplasmic MT complexes as visualized by indirect immunofluorescence microscopy and quantitated by direct binding of radiolabeled monoclonal antibody to cytoskeletons. The opposing effects of these drugs on MTs shows that there is a correlation between the extent of MT depolymerization and initiation of DNA synthesis. Initiation of DNA synthesis by colchicine in the presence of taxol is half-maximal when taxol and colchicine are added to cultures at a ratio of about 13 to 1. At this drug ratio, taxol stabilizes MTs near the nucleus, but MTs near the cell periphery are depolymerized. Maximal inhibition of DNA synthesis by taxol occurs only at taxol to colchicine ratios where MTs extend throughout the cytoplasm to the cell periphery. Thus, depolymerization of a small fraction of total MTs, particularly those near the periphery, may be sufficient to initiate proliferative events.