In the course of a systematic screening of sera from dogs suffering from autoimmune disorders, three sera were shown by indirect immunofluorescence to characteristically label the nucleoli and nucleoplasm of human cell lines (Hep-2 and HeLa). This pattern of staining persisted throughout the cell cycle, except for mitosis when the fluorescence was localized in extrachromosomal areas. By immunoblotting nuclear and subnuclear fractions, three polypeptides of 110,000, 95,000, and 45,000 Da apparent molecular weight were identified, which reacted with all three sera. By means of affinity purification, it was shown that an antibody specific for any one of the three proteins also reacts with the two others. This antigenic cross-reactivity suggested regions of structural homology shared by the three proteins. Indeed, treatment of nucleoli with high concentrations of DNase I containing residual proteolytic activity resulted in the disappearance of the 110- and 95-kDa proteins and the concomitant appearance of a doublet of 45-kDa proteins. Subnuclear localization studies indicated that all three polypeptides were located in both nucleoli and nucleoplasm. Significantly, the 110-kDa protein differs from the major nucleolar protein, nucleolin, by its electrophoretic mobility in two-dimensional gels, its location in nucleoli and in nucleoplasm, its absence in nucleolar organizer regions of chromosomes, and its differential solubility of DNase I. Therefore, the three antigenically related species reported in this study constitute a new class of nucleolar proteins.