We report here a mammalian cell-free system that can support chromatin assembly. The Xenopus laevis oocyte extract is the most extensively studied physiological system available for in vitro chromatin assembly. The lack of an appropriate mammalian cell-free system has forced investigators to transfer chromatin assembled in Xenopus extracts to a homologous mammalian cell extract to achieve RNA polymerase II (pol II)-specific transcription (33,42,65). The potential limitations of such heterologous systems underscore the need to develop mammalian cell-free systems that can support physiological chromatin assembly and pol 1I-mediated transcription of homologous genes.Numerous previous in vitro nucleosome assembly studies involved either reconstitution of purified core histones and naked DNA (24,25,54) or assembly of DNA in cell extracts (25,35,44,45,52