We report the successful establishment of 16 stable murine hybridoma monoclones that produce homogeneous antibodies against KB cell DNA polymerase alpha. All of the antibodies exhibit specific binding of polymerase alpha activity, and 3 of them possess anti-polymerase alpha neutralizing activity. None of the antibodies interacts detectably with KB cell DNA polymerases beta or gamma. All of the 5 antibodies so far examined demonstrate linear Scatchard binding plots and very high binding affinities, with equilibrium dissociation constants (Kd) ranging between 3.2 x 10(-9) and 3.4 x 10(-10) M. These monoclonal antibodies comprise a set of powerful and specific reagents that should facilitate the development and application of novel approaches to the complex biochemical mechanisms of mammalian DNA replication.