Abstract A comparative study was made on the pancreatic and T1 RNase digestion products of Novikoff hepatoma 28 S ribosomal RNA and its nucleolar precursors, i.e. nucleolar 28 S, 35 S, and 45 S RNA. No significant differences were found in the distribution of 32P in oligonucleotides obtained by pancreatic digestion of 28 S ribosomal RNA and nucleolar 28 S, 35 S, and 45 S RNA. Moreover, no discernible differences were noted in the electrophoretic mobilities of the large fragments obtained from 28 S rRNA, nucleolar 28 S, 35 S, and 45 S RNA after limited T1 RNase digestion. An electrophoretically homogeneous fragment referred to as the B3a fragment was obtained from the T1 RNase digest of ribosomal 28 S RNA and nucleolar 28 S, 35 S, and 45 S RNA. The B3a fragment contains 81% guanylic and cytidylic acid residues and has an approximate molecular weight of 200,000 and a Tm of 75°. On further hydrolysis by T1 RNase, smaller products of the B3a fragment were produced; one of these, the B4 fragment, which has an approximate molecular weight of 150,000, was obtained in an electrophoretically homogeneous state. This distribution of 32P in oligonucleotides of the B3a fragment of 28 S rRNA and nucleolar 28 S RNA was essentially the same. For other fragments, including the B4 fragment and the double band containing B3a and B3b, the oligonucleotide frequencies were essentially the same for 28 S ribosomal RNA and nucleolar 28 S, 35 S, and 45 S RNA.