Antisera to nucleoli of Novikoff hepatoma ascites and normal rat liver cells were produced in rabbits by injection of whole, isolated nucleoli. These antisera have been used to compare the nucleolar antigens that were partially fractionated by differential solubilization from nucleoli. Fourteen antigens were detected by these antisera; ten of these antigens were detected by both antisera. Ouchterlony double diffusion analysis of soluble extracts from normal rat liver and Novikoff hepatoma ascites nucleoli and fetal rat liver nuclei provided evidence for antigens found only in liver extracts, only in tumor extracts, or only in tumor and fetal extracts. Antisera preabsorbed to remove antibodies to common antigens of liver and tumor provided confirmatory evidence for one nucleolar antigen in liver that was not found in tumor or fetal rat liver, one antigen in tumor that was not found in adult or fetal rat liver, and three antigens in both tumor and fetal rat liver that were not found in adult rat liver. In addition, the antitumor nucleolar antiserum preabsorbed with liver nuclear extracts still produced positive nucleolar fluorescence in Novikoff hepatoma ascites cells but not in liver cells. Conversely, anti-liver nucleolar antiserum preabsorbed with tumor nucleolar extracts did not produce detectable tumor nucleolar fluorescence but did produce positive fluorescence in liver nucleoli.