We have shown that calf thymus DNA polymerase alpha-DNA primase complex (pol alpha-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. Here we tested the association of pol alpha-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with pol alpha-primase using anti-pol alpha antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by pol alpha-primase demonstrated that the interaction of pol alpha-primase with SVG was much weaker than that with CTPPS-1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind pol alpha-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to pol alpha-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, pol alpha-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, pol alpha-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows pol alpha-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.