Topoisomerase II (topo II) is an essential enzyme that alters DNA topology. This activity is important for a variety of chromosome functions including DNA replication, transcription, recombination, and chromosome condensation and segregation. Previously we localized topo II in mouse gametes and preimplantation embryos using isoform-specific antibodies demonstrating the presence of the enzyme in oocytes and embryos, but not sperm. To probe functions of topo II during preimplantation development, we treated mouse zygotes with 100 nM teniposide, and assessed embryo morphology and DNA replication. Teniposide blocked cleavage in 69% embryos; the remainder cleaved once but had abnormal nuclei. Teniposide-treated embryos were devoid of topo II immunofluorescence. Teniposide also prevented DNA replication, implicating topo II in this process. Embryos treated with a 2 hr pulse of teniposide recovered and developed to the blastocyst stage, indicating 100 nM teniposide did not induce apoptosis. To more specifically analyze topo IIalpha function, we treated zygotes with topo IIalpha-targeted antisense oligodeoxynucleotides. Most zygotes arrested at the 2-cell stage while controls developed into blastocysts indicating topo IIalpha is essential for preimplantation development. The absence of topo IIalpha, but not beta immunofluorescence in antisense-treated embryos confirms the specificity and impact of the treatment. In addition, topo IIalpha is newly synthesized at the 2-cell stage. These results establish an essential function for topo II in mouse preimplantation embryonic development.