Mouse L-cell nucleoli were isolated from sonicated nuclei by centrifugation and extensively treated with pancreatic DNase or micrococcal nuclease to obtain "core nucleoli." Core nucleoli still contained the precursors to rRNA and about 1% of the total nuclear DNA, which remained tightly bound even after the removal of some chromatin proteins with 2 M NaCl. The core nucleolar DNA electrophoresed in a series of discrete bands, 20 to about 200 base pairs in length. Hybridization tests with specific DNA probes showed that the DNA was devoid of sequences complementary to mouse sateilite, mouse Alu-like, and 5S RNA sequences. It also lacked sequences coding for cytoplasmic rRNA species, since it did not hybridize to the 18S to 28S portion of rDNA in Northern blot analyses and none of it was protected by hybridization to a 100-fold excess of total cytoplasmic RNA in S1 nuclease assays. However, the core nucleolar DNA did hybridize to nontranscribed and external transcribed spacer rDNA sequences. We infer that specific portions of rDNA are protected from DNase action by a tight association with nucleolar structural proteins.Because nucleoli show a structural arrangement which can be cytologically correlated with events in ribosome formation (9), their molecular organization is of great interest. The extensive studies of Busch and co-workers have characterized DNA fragments probably unique to nucleoli (10), preribosomes, and the nucleolar-specific U3 small nuclear RNA (snRNA) (8). However, nucleoli prepared by conventional methods (4, 6, 23) also include up to 50% of the nuclear DNA and its associated proteins (4, 6, 23). Since only about 0.1 to 0.3% of the nuclear DNA is rDNA, these preparations contain a great deal of other chromatin and nuclear material. Thus nucleolar-specific DNA, RNA, or protein species are seen against a very high background.We have used extensive treatment of nucleoli with DNase I or micrococcal nuclease to try to remove perinuclear or adventitiously associated chromatin and nucleoplasm selectively. We will report here that the resulting "core" fraction retains the preribosomes and snRNAs characteristic of nucleoli but now contains only a small fraction of the cellular DNA in a form highly protected against nuclease action. The DNA shows a characteristic size distribution and a restricted sequence content, including only spacer sequences of rDNA. Nuclei were collected by centrifugation through 0.5 M sucrose in buffer B (10 mM Tris-chloride [pH 7.4], 10 mM MgC12, 150 mM NaCl) at 800 x g for 10 min. They were then resuspended in buffer B (107/ml) and disrupted by sonication (50 W, 4 x 30 s, with a Branson Sonifier). A 20-,ug portion of DNase I per 107 nuclei (55 U/107 nuclei per ml) was added, and the extract was incubated at 37°C as indicated in the text. Nucleoli were isolated by centrifugation through a 10-ml sucrose step gradient containing 5 ml each of 0.25 and 0.88 M sucrose at 800 x g for 15 min and suspended in 1 mM Tris-chloride (pH 8.0)-i mM EDTA on ice for 15 min with occasional agitatio...