DNA synthesis in vitro was studied in rat giant trophoblast nuclei from parietal yolk sacs at Day 13 of development. These nuclei synthesize DNA by a series of endoreduplications in the absence of mitosis. Although the nuclei contain DNA polymerase alpha and beta activities, polymerase alpha activity is not expressed since DNA synthesis is insensitive to aphidicolin. In contrast, N-ethylmaleimide and the DNA polymerase beta inhibitors, 50 mM potassium phosphate, and 2',3'-dideoxythymidine triphosphate inhibit DNA synthesis in isolated nuclei to a degree identical with that seen with partially purified trophoblast DNA polymerase beta, suggesting that polymerase beta is the major polymerase activity in Day 13 trophoblast nuclei. DNA synthesis by these nuclei is dependent on ATP and inhibited by novobiocin and has been shown to be replicative rather than repair-type synthesis, since the newly synthesized DNA is contiguous with DNA replicated in vivo in the presence of a bromodeoxyuridine density label. The results suggest that DNA endoreduplication in these nuclei is being carried out by DNA polymerase beta.