The mechanism for incorporation of aphidicolin-sensitive DNA polymerase into reconstituting sperm nuclei was studied in a Xenopus egg extract cell-free system. Aphidicolin-sensitive DNA polymerase activity was sedimented along with the light membrane fraction of Xenopus egg extract on a discontinuous sucrose gradient. Treatment of the egg extract with Triton X-100 caused DNA polymerase activity to migrate to a lighter density position at which free proteins were distributed. DNA polymerase activity was incorporated into the reconstituting sperm nuclei from the egg extract, but no nuclear incorporation was observed in nuclei incubated in egg extracts which had been treated with Triton X-100 or sonicated. The incorporation was also prohibited by several different treatments of the egg extract resulting in incomplete assembly of the nuclear membrane on the sperm nuclei. On the other hand, there was no inhibition of nuclear incorporation into the sperm nuclei reconstituting in the extracts which had been depleted of WGA-binding pore complex proteins or which contained a specific inhibitor of topoisomerase II (ICRF-193). In these two cases, the nuclear double-layered membrane assembled normally, although in the former case the sperm nuclei lacked lamina and did not initiate DNA replication, and in the latter case the sperm nuclei did not decondense but initiated DNA replication. Thus, it is concluded that DNA polymerase activity is incorporated into the reconstituting nuclei via the membraneous/particulate fraction of the egg extract simultaneously with nuclear double-layered membrane assembly. The lamina assembly and the transport system via the nuclear envelope pore complex are suggested not to participate in DNA polymerase nuclear incorporation.