Tumor nucleoli were treated with polyclonal antisera to normal human tissue nucleoli to block some determinants common to tumor and normal tissue nucleoli. Immunization of mice with these immune complexes resulted in the development of a monoclonal antibody (FB2) to a novel Mr 120,000 nucleolar proliferation-associated antigen. By indirect immunofluorescence, antibody FB2 produced bright nucleolar staining in a variety of malignant tumors, including cancers of the breast, liver, gastrointestinal tract, genitourinary tract, blood, lymph system, lung, and brain. Although specific nucleolar immunofluorescence was not detectable in most normal tissues, it was detectable in some proliferating nonmalignant tissues including spermatogonia of the testes, ductal regions of hypertrophied prostates, and phytohemagglutinin-stimulated lymphocytes. The Mr 120,000 antigen was not detectable in 48-h serum-deprived HeLa cells but was readily detectable (within 30 min) following serum refeeding. The Mr 120,000 antigen was not detected in retinoic acid-treated HL-60 cells following morphological differentiation but was detectable in 48-h phytohemagglutinin-treated lymphocytes. These studies suggest that the Mr 120,000 antigen is a proliferation-associated antigen which plays a role in the early G1 phase of the cell cycle.