Nascent DNA chains of papova viruses and animal cells are transiently linked to a 5′-terminal oligoribonucleotide, termed initiator RNA, which is implicated in their priming. To study the biosynthesis and degradation of iRNA† † Abbreviations used: iRNA, initiator RNA; RI DNA, replicative intermediate DNA; HEPES, N-2-hydroxyethyl piperazine-N′-2-ethanesulfonic acid; EGTA, ethyleneglycol-bis(β-aminoethyl ether)-N,N′-tetraacetic acid., as well as its location within replicating DNA, iRNA was labeled in isolated nuclei of simian virus 40 (SV40)-infected monkey kidney cells with rNTP precursors, [α-32P]rCTP or [β-32P]rGTP. Incubation in the presence of up to 200 μg α-amanitin/ml served to reduce non-iRNA transcription, although it did not affect the synthesis of iRNA. This result also confirmed that RNA polymerases II and III are not involved in iRNA synthesis. Replicative intermediate SV40 DNA was then purified by salt/ detergent extraction and gel filtration and treated with pancreatic RNAase to remove residual non-iRNA-bound 32P. Initiator RNA was not affected, however, suggesting that it was entirely base-paired to the parental template. Denaturation of the purified SV40 RI DNA, followed by gel filtration, resolved the nascent DNA into short chains (Okazaki pieces) and long chains, iRNA being associated mainly with the former. Upon DNAase I digestion, both short and long chains yielded intact iRNA, migrating on the gel in the (pA)9-12 marker range. However, the long chains yielded in addition shorter oligonucleotides, apparently partially degraded iRNA. Further enzymic digestions of iRNA labeled either with [α-32P]rCTP or [β-32P]rGTP indicated that a fraction of it contained a 5′-terminal triphosphate group. The retrograde fork polarity of iRNA-containing Okazaki pieces was demonstrated by their highly asymmetric hybridization to single-stranded DNA probes of unidirectional replication fork polarities, as previously seen with Okazaki pieces labeled in their DNA moiety. In contrast, iRNA-tagged long chains hybridized evenly with probes of both fork polarities, indicating that Okazaki pieces become ligated to the continuously growing strand of the opposite fork early in replication.