In ATT, a human autoimmune serum, we found anti-nucleolar antibodies that recognized nucleolar antigens confined to a single nucleolar compartment, the dense fibrillar component (DFC). We localized these antigens by immunoelectron microscopy in DFC of HeLa cell nucleoli both on Lowicryl sections and cryoultrathin sections without embedding. The antigens were solubilized by incubation with 2M NaCl but not by RNase or DNase treatment. The ATT serum crossreacted with rat liver nucleoli and PtK1 cell nucleoli in which immunofluorescence labelling displayed a clumpy pattern. During mitosis, the antigens dispersed in the cytoplasm until late telophase, when they gathered in the prenucleolar bodies. In human peripheral lymphocytes, or HeLa cells treated with actinomycin D, the antigens were still present but the fluorescence intensity decreased. By immunoblotting using human nuclear extracts, the ATT serum bound to a 116,000 Mr protein at dilutions up to 1:2000. The reactivity of this band diminished with actinomycin D-treated nuclear extracts. Two minor bands were also observed at 97 and 70K (K = 10(3) Mr). Immunopurification by competition or elution demonstrated that the 116K antigens were at the origin of the nucleolar labelling. This DFC marker appeared to be different from the NOR-silver-stained proteins, which in our preparations exhibited apparent molecular weights of 105, 80 and 38-40K. In addition, these 116K antigens did not exhibit the characteristics described for DNA topoisomerase I, fibrillarin or nucleolin. We propose the 116K antigen as a new marker of the DFC of the nucleoli.