SV40 DNA, replicating in isolated nuclei, contains RNA-DNA covalent linkages which were quantitated by measuring the release of [2′(3′)-32P]rNMPs from [32P]-DNA incubated in KOH (32P-label transfer assay). More than 96% of the 32P label released during this incubation was shown to be in [2′(3,)-32P]rNMPs by chemically converting it into cyclic [2,:3′-32P]rNMPs and then enzymatically cleaving the cyclic nucleotides to produce [3′-32P]rNMPs. [α-32P]dNTP, incorporated into DNA, was identified as the 32P donor because the amount of 32P-label transferred was proportional to the specific radioactivity of the labeled substrate. All 16 possible rN-dN linkages were found in SV40 replicating DNA at frequencies that suggested a near-random distribution on the genome. These RNA-DNA covalent linkages behaved as transient intermediates in DNA synthesis; they disappeared at the same rate that nascent 4S DNA chains (“Okazaki pieces”) were joined to the growing daughter strands. Therefore, these linkages exhibited kinetic properties consistent with the proposed role of RNA as a primer for discontinuous DNA synthesis. When 4S DNA joining was inhibited by the absence of cytosol, the disappearance of RNA-DNA covalent linkages was not prevented. Inhibition of DNA synthesis with either ara-C TP or ara-ATP also failed to block the removal of RNA-DNA covalent linkages. Thus, the excision of these putative RNA primers does not appear to require either the concomitant joining of 4S DNA chains or DNA synthesis.