Evidence was obtained for tight association of DNA primase activity with a subspecies of mouse DNA polymerase alpha by study with immunoadsorption assay using two monoclonal antibodies specific for human DNA polymerase alpha that have been shown to react with mouse murine myeloma DNA polymerase alpha (Tanaka, S., Hu, S.-Z., Wang, T.-S.-F. & Korn, D. (1982) J. Biol. Chem. 257, 8386-8390). This result was supported by the finding that ethidium bromide at concentrations of less than 20 microM somewhat stimulated the syntheses of DNA and initiator RNA on unprimed poly(dT) by the novel subspecies of DNA polymerase alpha, but strongly inhibited DNA synthesis with poly(dT) X oligo(rA), suggesting that the conversion of synthesis from initiator RNA to DNA is continuous. Furthermore, the results of neutralization assay with the antibodies and experiment using aphidicolin suggested that the primase site is functionally distinguishable from the catalytic site of DNA polymerase activity.