In the S-phase of the eukaryotic cell cycle, newly replicated DNA is assembled into chromatin. I used indirect immunofluorescence microscopy to localize the sites of chromatin assembly in respect to DNA replication. Replication foci in the nuclei of permeabilized HeLa cells were labeled by incorporation of biotin-16-dUTP and detected by fluorescent streptavidin. Prelabeling of replication foci in vivo with bromodeoxyuridine showed that replication in permeabilized cells proceeds at preexisting replication forks. The localization of chromatin assembly factor 1 (CAF-1) was determined with subunit-specific monoclonal antibodies. CAF-1 is not detectable in mitotic cells and is detectable only at background levels in about 60% of all interphase nuclei. The other interphase nuclei show an intense punctate immunostaining of CAF-1. These sites of CAF-1 colocalize with replication foci during all stages of the S-phase. No other discrete sites of CAF-1 are observed. Human replication protein A (RPA) colocalizes with these replication/chromatin assembly sites. In addition, extra nuclear sites of RPA are observed that probably represent prereplication foci, poised for initiation of DNA replication.