We have successfully established 16 stable murine hybridomas that secrete monoclonal antibodies specific for human DNA polymerase alpha. The results of immunocytochemical studies, using 4 of these monoclonal antibodies and immunoperoxidase detection methods, document the exclusively intranuclear localization of DNA polymerase alpha in three separate lines of cultured human cells. By light microscopy, the immunoperoxidase reaction product exhibits a diffuse pattern of distribution within the nucleoplasm, but nucleoli are clearly negative. In cultures of the transformed lines, KB nd BeWo, more than 955 of the cells are positive, suggesting that intranuclear DNA polymerase alpha antigens persist throughout the mitotic cycle. In striking contrast, in the normal diploid fibroblast line, WI-38, a smaller fraction of the cultured cells is positive, and there is no detectable polymerase alpha antigen in the closely apposed cells of microcolonies that are presumed to be contact-arrested and no longer mitotically cycling. In cells in mitosis that have dissolved their nuclear envelopes (and are thus transiently anucleate), the anti-polymerase alpha reaction continues to be strongly positive, and in this single circumstance the reaction product is diffusely distributed throughout the cell cytoplasm. Initial electron microscopic examination of KB cells confirms and extends these observations. The immunoperoxidase reaction product is essentially limited to the nuclear compartment and is predominantly distributed in the midzonal region of the nucleoplasm between centrally disposed nucleoli and peripherally located blocks of condensed chromatin.