The yeast nucleolar protein Rrp5p is the only known trans-acting factor that is essential for the synthesis of both 18S rRNA and the major, short form of 5.8S (5.8S(S)) rRNA, which were thought to be produced in two independent sets of pre-rRNA processing reactions. To identify domains within Rrp5p required for either processing pathway, we have analyzed a set of eight deletion mutants that together cover the entire RRP5 sequence. Surprisingly, only one of the deletions is lethal, indicating that regions encompassing about 80% of the protein can be removed individually without disrupting its essential biological function. Biochemical analysis clearly demonstrated the presence of two distinct functional domains. Removal of each of three contiguous segments from the N-terminal half specifically inhibits the formation of 5.8S(S) rRNA, whereas deleting part of the C-terminal region of the protein only blocks the production of 18S rRNA. The latter phenotype is also caused by a temperature-sensitive mutation within the same C-terminal region. The two functional regions identified by the mutational analysis appear to be correlated with the structural domains detected by computer analysis. They can be physically separated, as demonstrated by the fact that full Rrp5p activity can be supplied by two contiguous protein fragments expressed in trans.