An in vitro HeLa chromatin replication system was developed and characterized. Purified nuclei synthesized DNA from replication forks which were undergoing chain elongation at the time of cell fractionation. DNA replication in isolated nuclei was deficient in DNA maturation functions; the Okazaki fragments synthesized in vitro accumulated and were ligated to high-molecular-weight DNA with low efficiency. The chromatin protein components of nuclei incubated in the DNA replication assay conditions were investigated for displacement, degradation, and exchange. Displacement of total nuclear protein during in vitro incubation occurred to the extent of 3%, and involved only the nonhistone nuclear proteins. Degradation was not detectable, assayed both by loss of acid-soluble radioactivity and by protein electrophoretic patterns in polyacrylamide gels. No detectable protein was exchanged from chromatin to exogenous DNA.