We have developed a system for mutational analysis of Saccharomyces cerevisiae ribosomal RNA in vivo in which yeast cells can be made completely dependent on mutant rRNA and ribosomes by a simple switch in carbon source. The system is based on a yeast strain defective in RNA polymerase I (Pol I) transcription [Nogi et al. (1991) Proc. Natl. Acad. Sci. USA 88, 3962-3966]. This normally inviable strain was rescued by integration of multiple copies of the complete 37S pre-rRNA operon under control of the inducible, Pol II-transcribed GAL7 promoter into the rDNA repeat on chromosome XII. The resulting YJV100 strain can only grow on medium containing galactose as the carbon source. A second, episomal vector was constructed in which the rDNA unit was placed under control of the constitutive PGK1 promoter. YJV100 cells transformed with this vector are now also able to grow on glucose-based medium making the cells completely dependent on plasmid-encoded rRNA. We show that the Pol II-transcribed pre-rRNA is processed and assembled similarly to authentic Pol I-synthesised pre-rRNA, making this 'in vivo Pol II system' suitable for the detailed analysis of rRNA mutations, even highly deleterious ones, affecting ribosome biogenesis or function. A clear demonstration of this is our finding that an insertion into variable region V8 in 17S rRNA, previously judged to be neutral with respect to processing of 17S rRNA, its assembly into 40S subunits and the polysomal distribution of these subunits [Musters et al. (1989), Mol. Cell. Biol. 9, 551-559], is in fact a lethal mutation.