Slx9p (Ygr081cp) is a nonessential yeast protein previously linked genetically with the DNA helicase Sgs1p. Here we report that Slx9p is involved in ribosome biogenesis in the yeast Saccharomyces cerevisiae. Deletion of SLX9 results in a mild growth defect and a reduction in the level of 18S rRNA. Co-immunoprecipitation experiments showed that Slx9p is associated with 35S, 23S, and 20S pre-rRNA, as well as U3 snoRNA and, thus, is a bona fide component of pre-ribosomes. The most striking effects on pre-rRNA processing resulting from deletion of SLX9 is the accumulation of the mutually exclusive 21S and 27SA2 pre-rRNA. Furthermore, deletion of SLX9 is synthetically lethal with mutations in Rrp5p that block cleavage at either site A2 or A3. We conclude that Slx9p has a unique role in the processing events responsible for separating the 66S and 43S pre-ribosomal particles. Interestingly, homologs of Slx9p were found only in other yeast species, indicating that the protein has been considerably less well conserved during evolution than the majority of trans-acting processing factors. Published by Cold Spring Harbor Laboratory Press. Copyright © 2006 RNA Society.