When rRNA (ribosomal ribonucleates) or sRNA (soluble ribonucleates) are hydrolyzed in alkali, the resulting hydrolysis products can be resolved into four separate classes: nucleosides (N), nucleoside 2′-and 3′-phosphates (Np), nucleoside 2′(3′),5′-diphosphates (pNp), and alkali-stable dinucleotides (NxpNp, where x denotes 2′-O-methylation). The N and pNp compounds derive from 5′-linked and 3′-linked termini, respectively, while Np and NxpNp compounds derive from internal positions of polynucleotide chains: (3′-linked terminus) pNpNpNpN·····pNpNpNpN (5′-linked terminus). In the present study, the distribution of radioactivity among these different classes of compounds in alkali hydrolysates of L cell rRNA, has been compared with corresponding data for rapidly labeled RNA from L cells. Three rapidly labeled RNA specimens were prepared from L cells that had been exposed to four tritiated nucleosides (adenosine (A), guanosine (G), cytidine (C), and uridine (U)) for 15, 30, and 90 min, while rRNA was prepared from cells exposed to the tritiated nucleosides for 24 hr. All RNA preparations were repeatedly precipitated from 2.5 M sodium chloride solution at 0°, in order to remove sRNA. The rapidly labeled RNA preparations were polydisperse and characterized by different amounts (20-75%) of material sedimenting faster than 28 S, whereas the 24-hr preparation displayed an essentially bimodal sedimentation profile, with peaks at 16 and 28 S. In the case of nucleosides, all preparations were remarkably similar with respect to the proportionate amount of radioactivity in a given nucleoside (N) relative to its homologous nucleotide (Np), i.e., A/Ap, G/Gp, C/Cp, and U/Up radioactivity ratios were similar for all preparations. By contrast, in the case of pNp and NxpNp compounds, the proportionate amounts of radioactivity in rapidly labeled RNA were much lower than for rRNA, after 15 min, but approached the proportions for rRNA, after 90 min. These empirical similarities and differences are based on the primary experimental measurements, no assumptions having been made with respect to the relative mean specific activities at terminal and nonterminal positions in the polynucleotide chains of the different preparations. As such the relations can be considered as characteristic analytical parameters, which distinguish rRNA from rapidly labeled RNA. By invoking assumptions, the primary data can be used to assess molar percentages of the different components in the RNA preparations. These data are discussed in terms of the degree to which they might reflect features of the primary structure of the polynucleotide chains, and also in terms of the extent to which they might reflect differences of mean specific activity between the termini of the polynucleotide chains in the different RNA specimens.