A nucleolar chromatin antigen (NoAg-1) found in Novikoff hepatoma but not in normal liver has been purified to homogeneity as shown by two-dimensional gel electrophoresis. Initial purification of NoAg-1 was partially achieved by isolation of nucleolar chromatin and fractionation of its proteins by successive extraction with solutions of increasing salt concentration. Further purification of this antigen was achieved by affinity and hydroxylapatite chromatography. Although approximately 50% of the NoAg-1 antigen was in the 0.6 M NaCl extract of Novikoff nucleoli, it was less pure than in the 2 M NaCl:5 M urea extract which contained 25% of the NoAg-1 at a purity of 40%. The highly purified NoAg-1 had an approximate molecular weight of 60,000 and pl of 5.1; the yield of NoAg-1 was 0.22% of the total nucleolar proteins.