Abstract Nucleoli were prepared from rat liver nuclei isolated in 2.2 m sucrose. Assays for glutamate dehydrogenase, adenylate kinase, catalase, acid phosphatase, 5'-nucleotidase, glucose 6-phosphatase, lactate dehydrogenase, and pyruvate kinase as markers for cytoplasmic contamination revealed that the isolated nucleoli were in a high state of purity. Studies of the intranuclear distribution pattern of the enzymes and determination of the specific activities of nuclear enzymes showed that ribonucleic acid polymerase, ribonuclease, and, to a lesser extent, diphosphopyridine nucleotide pyrophosphorylase and adenosine triphosphatase A are localized to the nucleolus. Nuclear acid and alkaline deoxyribonuclease and adenosine triphosphatase B were not found to be localized to the nucleolus. The increased ribonuclease activity produced by sonic oscillation, Triton X-100, and p-chloromercuribenzoate, as well as the different response to these agents of nuclear fractions from normal rats and from rats treated with thioacetamide or actinomycin D, or both, shows that there may be more than one mechanism of latency of nuclear and nucleolar ribonucleases. The differences in substrate specificities of ribonuclease, adenosine triphosphatase A, and adenosine triphosphatase B of nuclear and of nucleolar origin point to qualitative differences between these nuclear and nucleolar enzymes. The existence of a polynucleotide phosphorylase activity was shown by the formation of nucleoside diphosphates from polyribonucleotides when the latter were incubated with nuclear subfractions.