De novo DNA synthesis on poly(dT) by a novel mouse DNA polymerase, here named "DNA replicase," was examined for the synthesis of RNA which functions as a primer in the subsequent synthesis of DNA. As has been reported previously (Yagura, T., Kozu, T., and Seno, T. (1982) J. Biochem. (Tokyo) 91, 607-618), a novel RNA polymerase activity, which is distinguished from those of classical RNA polymerases, is associated with DNA replicase. The synthesis of RNA and DNA by DNA replicase (Mr = 16 X 10(4), by glycerol gradient sedimentation analysis) was greatly stimulated by a specific stimulating factor (Mr = 13 X 10(4), by glycerol gradient sedimentation analysis) which was found to consist of two subunits (Mr = 63 X 10(3), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis). Nearest neighbor analysis in which transfer of 32P from alpha-labeled nucleoside triphosphates to ribo- and deoxyribonucleotides was examined, showed th at RNA of 8-10 nucleotides long was covalently linked to the 5'-end of the DNA product molecule. This RNA, named initiator RNA, had a triphosphate group at its 5' terminus and its size and synthesis were little affected by the addition of high concentrations of deoxynucleoside triphosphate, while in these conditions deoxyribonucleotides were incorporated into initiator RNA to a limited extent. The characteristics of the DNA replicase and stimulating factor that cooperate to synthesize initiator RNA for subsequent DNA synthesis on single-stranded DNA are important because these components seem to be involved in a reaction required to initiate the synthesis of discontinuous earliest DNA intermediates (Okazaki fragments) in chromosomal DNA replication of eukaryotic cells.