Nucleolar chromatin of Novikoff hepatoma ascites cells contains an antigen (no-Ag1) detected with antinucleolar antibodies by the immunodiffusion technique. This antigen was distinguished from the previously reported nuclear chromatin antigen NAg-1 (19) by the findings that tumor nucleolar antibodies which formed immunoprecipitin bands with no-Ag1 did not do so with NAg-1 and that tumor cytosol, which contains NAg-1, formed immunoprecipitin bands with tumor chromatin antibodies but not with antibodies to tumor nucleoli. Tumor nucleolar chromatin contains both NAg-1 and no-Ag1, but only no-Ag1 formed bands with tumor nucleolar antibodies, no-Ag1 is a component of tumor nucleolar chromatin that was not soluble in 0.075 M NaCl-0.025 M EDTA, pH 8, and only slightly soluble in 0.01 M Tris-HCl, pH 8. NO-Ag1 was not found in liver nucleoli. Antibodies to liver nucleoli formed immunoprecipitin bands with liver nucleolar antigens but none were confluent with those formed between tumor nucleolar antibodies and antigens of tumor nucleolar chromatin. Absorption of the tumor nucleolar antibodies with whole tumor cells or whole liver pressate did not alter band formation with no-Ag1. Three antigens in liver nucleoli were not found in tumor nucleoli.