Using in vitro replication assays, we compared native with salt-treated simian virus 40 minichromosomes isolated from infected cell nuclei. Minichromosomes from both preparations contain the full complement of nucleosomes, but salt treatment removes histone H1 and a fraction of nonhistone chromatin proteins. Both types of minichromosomes served well as templates for in vitro replication, but the structures of the replication products were strikingly different. Replicated salt-treated minichromosomes contained, on average, about half the normal number of nucleosomes as previously shown (T. Krude and R. Knippers, Mol. Cell. Biol. 11:6257-6267, 1991). In contrast, the replicated untreated minichromosomes were found to be densely packed with nucleosomes, indicating that an assembly of new nucleosomes occurred during in vitro replication. Biochemical and immunological data showed that the fraction of nonhistone chromatin proteins associated with native minichromosomes includes a nucleosome assembly activity that appears to be closely related to chromatin assembly factor I (S. Smith and B. W. Stillman, Cell 58:15-25, 1989). Furthermore, this minichromosome-bound nucleosome assembly factor is able to exert its activity in trans to replicating protein-free competitor DNA. Thus, native chromatin itself contains the activities required for an ordered assembly of nucleosomes during the replication process.