With a specific stimulating factor of mouse DNA replicase for its detection, a novel form of DNA polymerase alpha (DNA replicase) associated with DNA primase activity was partially purified from several vertebrates, i.e. the cherry salmon Oncorhyncus masou, the frog Xenopus laevis, the chick, and human (HeLa cells). Activity similar to DNA replicase was also partially purified from embryos of the sea urchin Anthocidaris crassispina. In all vertebrates examined, two forms of DNA polymerase alpha were separated by chromatography on ion-exchange columns; one form (DNA replicase) was associated with DNA primase activity and could utilize unprimed single-stranded DNAs as template, and the other could not utilize unprimed single-stranded DNAs. The sedimentation coefficient of the former, the novel form, obtained from each vertebrate in a glycerol gradient at high ionic strength was slightly larger than that of the other form which had no primase activity, except in the case of chick embryos where the sedimentation coefficients of the two forms were almost the same. The initiator RNA synthesized with the DNA primase activity associated with DNA replicase obtained from salmon, chick, HeLa cells, and sea urchin was 8 to 10 nucleotides long. The stimulating factor obtained from Ehrlich ascites cells has been found to stimulate both the activities of DNA primase and DNA polymerase in DNA replicase obtained from all the vertebrates examined, when unprimed single-stranded DNA was used as template, while the factor failed to stimulate both the activities of the enzyme of sea urchin embryos. This factor thus should be an effective tool in studies on the mechanism of vertebrate DNA replication.