Isolated uterine nuclei incorporate [3H]deoxythymidine-5'-triphosphate into acid-precipitable material for 60 min. The rate and extent of incorporation is markedly enhanced by prior treatment of rats with estradiol-17 beta. This stimulation is dose dependent and follows a time course which parallels that which is observed in intact uteri. The maximum response occurs 24 h after an injection of estradiol-17 beta when the DNA synthesis rate has shown an 8.5 +/- 0.9-fold average stimulation over 34 experiments. When a small dose of estradiol-17 beta was injected directly into one uterine horn, DNA synthesis was stimulated in nuclei isolated from that horn but not in nuclei from the vehicle-injected contralateral horn. This suggests that the mitogenic effect of estrogen is direct and not mediated by systemic factors. As shown by diethylaminoethyl-cellulose chromatography and inhibition studies with N-ethylmaleimide and 2':3'-dioxythymidine-5'-triphosphate, the uterine nuclei contain both DNA polymerases alpha and beta; however, alpha-polymerase alone appears to be responsible for the estrogen-stimulated DNA synthesis. When soluble DNA polymerase alpha-activity and endogenous DNA synthesis rates were simultaneously measured, both were found to be initially stimulated between 12 and 15 h after an injection of estradiol-17 beta. However, because the fold-stimulation of alpha-polymerase activity was only half that of DNA synthesis, it appears that DNA synthesis is not merely a simple function of polymerase activity.