A recessive temperature sensitive mutant has been isolated that is defective in ribosomal RNA processing. By Northern analysis, this mutant was found to accumulate three novel rRNA species: 23S', 18S' and 7S', each of which contains sequences from the spacer region between 25S and 18S rRNA. 35S pre-rRNA accumulates, while the level of the 20S and 27S rRNA processing intermediates is depressed. Pulse-chase analysis demonstrates that the processing of 35S pre-rRNA is slowed. The defect in the mutant appears to be at the first processing step, which generates 20S and 27S rRNA. 7S' RNA is a form of 5.8S RNA whose 5' end is extended by 149 nucleotides to a position just 5 nucleotides downstream of the normal cleavage site that produces 20S and 27S rRNA. 7S' RNA can assemble into 60S ribosomal subunits, but such subunits are relatively ineffective in joining polyribosomes. A single lesion is responsible for the pre-rRNA processing defect and the temperature sensitivity. The affected gene is designated RRP2.