8 S RNA of Novikoff hepatoma was characterized by fingerprinting, sequencing gels, and by hybridization to rat ribosomal DNA clones. The data obtained show that 8 S RNA is 273 or 274 nucleotides long; ribosomal 5.8 S RNA is its 5'-terminal 156 nucleotides. All the post-transcriptional modifications found in 5.8 S rRNA were also found in 8 S RNA; no other modifications were found. The 3'-terminal 118 nucleotides were consistent with the adjoining internal transcribed spacer sequence in rDNA (Subrahmanyam, C. S., Cassidy, B., Busch, H., and Rothblum, L. (1982) Nucleic Acids Res. 10, 3667-3680). Based on its nucleolar localization, the finding that all the 8 S RNA is hydrogen-bonded to preribosomal RNA and its consistency in sequence to the cloned rat ribosomal DNA sequence, it appears that 8 S RNA is a relatively stable intermediate in the formation of 5.8 S rRNA from 45 S pre-rRNA. This stable intermediate RNA may be a useful substrate for studies on rRNA processing and for studies on eukaryotic rRNA-processing enzyme(s).