Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase α (pol α-primase) was compared with those synthesized by a multiprotein form of DNA polymerase α (pol α2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribo-nucleotide. Pol α2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol α-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vitro. Pol α2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol α-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase a affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.