During the nucleolar maturation of eukaryotic ribosomal RNAs, many selected uridines are converted into pseudouridine by a thus far undefined mechanism. The nucleolus contains a large number of small RNAs (snoRNAs) that share two conserved sequence elements, box H and ACA. In this study, we demonstrate that site-specific pseudouridylation of rRNAs relies on short ribosomal signal sequences that are complementary to sequences in box H/ACA snoRNAs. Genetic depletion and reconstitution studies on yeast snR5 and snR36 snoRNAs demonstrate that box H/ACA snoRNAs function as guide RNAs in rRNA pseudouridylation. These results define a novel function for snoRNAs and further reinforce the idea that base pairing is the most common way to obtain specific substrate-"enzyme" interactions during rRNA maturation.