The monoclonal antibody mAbH6, which recognizes one human Ku-protein (p70), cross-reacted with the counterpart protein from Xenopus eggs. The Xenopus antigen purified with a mAbH6-Sepharose column was a complex of 88 kDa and 72 kDa proteins. The role of Ku protein in nuclear structure formation was studied using a cell-free nuclear assembly extract derived from Xenopus interphase eggs. The protein was distributed on the surface of demembranated sperm chromatin and in the membrane vesicle fraction of the nuclear assembly extract. Addition of mAbH6 to the assembly extracts prevented the completion of reconstitution of pronuclei from demembranated sperm chromatins, although partial decondensation mediated by nucleoplasmin was not impeded. As a result, the sperm chromatin remained in partially swollen structures or formed round but small anomalous nuclei. Nuclear membranes were formed on the nuclei in mAbH6-inhibited extract systems, but DNA synthesis was largely decreased, suggesting the incomplete reconstitution of pronuclei. The incorporation of lamin to the nuclei was inhibited by mAbH6. It is suggested that the Xenopus Ku-homologous protein has a role in the formation of the lamin layer after nuclear membrane reconstitution.