Xenopus egg extracts, which support nuclear assembly and DNA replication in vitro, were physically depleted of lamin B3 using monoclonal antibodies linked to magnetic beads. Depleted extracts were still able to support nuclear envelope assembly around demembranated sperm heads but the resulting pronuclei lacked a lamina and were unable to initiate semiconservative DNA replication or to assemble replicases, confirming previous data. Immunoblotting analysis of isolated nuclei and nuclear matrix fractions indicated that lamin-depleted nuclei still accumulated nucleoporins and PCNA. Furthermore, the rate of PCNA uptake was identical in lamin-depleted and control nuclei. However, neither the nucleoporins nor the PCNA was associated with nuclear matrix fractions. The major protein components of sperm pronuclear matrix fractions were characterized by two-dimensional gel electrophoresis. Of these proteins only three out of 22 species, other than the lamins, were significantly reduced in lamin-depleted nuclei, indicating that these nuclei do assemble a nuclear matrix.