We report five novel monoclonal antibodies (Ki-S1, Ki-S4, Ki-S6, Ki-S7, and KiS8) reactive with a proliferation-related nuclear antigen. In immunoprecipitation and Western blot experiments using crude nuclear extracts, they recognized a protein of 170 kD that, after proteolytic digestion of the immunoprecipitate and sequencing of the resulting peptides, was identified as the α-isoform of human topoisomerase II. This was confirmed by testing the antibodies on a highly purified enzyme preparation. Crossreactivity with topoisomerase IIβ was ruled out by testing the antibodies on crude extracts from yeast cells expressing the β-isoform exclusively. The antibodies bind the antigen with different affinities and at different epitopes, apparently located within the carboxyl third of the enzyme. All five antibodies are suitable for archival material after adequate antigen retrieval, thereby enabling retrospective studies. This report illustrates the tissue and subcellular distribution of the antigen through the cell cycle by immunohistochemistry and confocal fluorescence microscopy. The antibodies will be useful tools in further analysis of morphological and functional aspects of topoisomerase II and may serve diagnostic purposes, as well as providing prognostic information in tumor pathology.