The human tumor nucleolar antigens found 1st in the HeLa [human cervical carcinoma] cells and subsequently in a broad range of human cancers were characterized. For visualization of the antigens, HeLa cell nucleolar or nuclear protein fractions were analyzed on 4% polyacrylamide isoelectric focusing gels. The gels were incubated with rabbit antisera to HeLa cell nucleoli and then with fluorescein- or peroxidase-conjugated goat anti-rabbit IgG. With this technique, 2 major nucleolar antigens (focusing at pH 6.3 and pH 6.1) were identified. These antigens were also found in the Namalwa cell, but not in human liver cells. Purification of the antigen(s) was achieved by selective extraction of Namalwa cell nuclei with 10 mM Tris-HCl (pH 8), 40-100% ammonium sulfate precipitation, diethylaminoethyl cellulose chromatography in which the antigen was eluted with 0.15 M NaCl buffer (DE-0.15 M fraction), and use of isoelectric focusing gels. The immunostained bands (HuAg 6.3 and 6.1) and the bright nucleolar immunofluorescence of the HeLa cells were not observed after the antisera were preabsorbed with the DE-0.15 M fraction. The immunostained bands (HuAg 6.3 and HuAg 6.1) and the nucleolar immunofluorescence of the HeLa cells were also observed when isoelectric focusing gels were incubated with antiserum from rabbits immunized with the DE-0.15 M fraction. On the sodium dodecyl sulfate second dimension of the 2-dimensional polyacrylamide gel electrophoresis, the antigen(s) migrated as single spots with apparent MW of 54,000.