Two-dimensional polyacrylamide gel electrophoresis patterns of 32P-labeled nuclear proteins in Novikoff hepatoma and regenerating liver relatively small number of the many nuclear protein spots were phosphorylated. The major phosphoprotein spots differed in size and shape from the stained protein spots. Seven phosphoproteins, 125/5.8, 125/7.2, 100/5.9, 85/5.9, 56/5.1, and 50/5.1 (mol. wt. x 10-(3)/pI in the Novikoff hepatoma nuclei were not found in the 18-h regenerating liver nuclei. One nuclear phosphoprotein, 54/6.5, was found in the liver but not in the hepatoma. The four major phosphoproteins, 45/5.3-6.0, 40/5.3-6.0, 35/5.3-6.0, and 25/5.8-6.8 containing about 60% of the total 32Pi of the nuclear proteins, were found in both types of cells. These proteins were of unusual density and shape. Some proteins, such as 125/5.8 and 85/5.9, were enriched in 0.01 M tris extract of the tumor, and proteins 125/7.2 and 56/6.1 were enriched in 0.35 M NaCl extract of the tumor.