The eukaryotic single-stranded DNA binding protein replication protein A (RPA) participates in major DNA transactions. RPA also interacts through its middle subunit (Rpa2) with regulators of the cell division cycle and of the response to DNA damage. A specific contact between Rpa2 and nascent simian virus 40 DNA was revealed by in situ UV cross-linking. The dynamic attributes of the cross-linked DNA, namely, its size distribution, RNA primer content, and replication fork polarity, were determined. These data suggest that Rpa2 contacts the early DNA chain intermediates synthesized by DNA polymerase α- primase (RNA-DNA primers) but not more advanced products. Possible signaling functions of Rpa2 are discussed, and current models of eukaryotic lagging- strand DNA synthesis are evaluated in view of our results.