Chromatin assembly factor I (CAF-I) is a multisubunit protein complex purified from the nuclei of human cells and required for chromatin assembly during DNA replication in vitro. Purified CAF-I promotes chromatin assembly in a reaction that is dependent upon, and coupled with, DNA replication and is therefore likely to reflect events that occur during S phase in vivo. In order to investigate the regulation and mechanism of CAF-I and the replication-dependent chromatin assembly process, we have used the purified protein to raise monoclonal antibodies. In this report we describe the characterization of a panel of monoclonal antibodies which recognize different subunits of the CAF-I complex. We use immunoprecipitation analysis to show that CAF-I exists as a multiprotein complex in vivo and that some of the polypeptides are phosphorylated. In addition, immunocytochemistry demonstrates that CAF-I is localized to the nucleus of human cells. Finally, monoclonal antibodies directed against the individual subunits of CAF-I immunodeplete chromatin assembly activity from nuclear extracts, confirming that CAF-I is a multisubunit protein required for chromatin assembly in vitro.