Protein B23 is a major RNA-associated nucleolar protein and putative ribosome assembly factor which exists in at least two isoforms designated B23.1 and B23.2. Recently, it has been reported that B23 is copurified with DNA polymerase alpha-primase complex. To examine its possible role in DNA replication, the effects of B23 on DNA polymerase activities were investigated. B23.1 purified from rat Novikoff hepatoma ascites cell nucleoli stimulated the activity of DNA polymerase alpha by as much as 3-to 4-fold in a dose-dependent manner, while it showed little effect on the activities of DNA polymerase beta, gamma, and primase. Rat recombinant B23.1 showed the same stimulation as that of B23.1 from Novikoff cells. In contrast, isoform B23.2 showed no effect on the activity of DNA polymerase alpha, suggesting that C-terminal region of B23.1 is important in its activity in the stimulation of DNA polymerase alpha.