Many or all of the sites of pseudouridine (Ψ) formation in eukaryotic rRNA are selected by site-specific base-pairing with members of the box H + ACA class of small nucleolar RNAs (snoRNAs). Database searches previously identified strong homology between the rat nucleolar protein Nap57p, its yeast homolog Cbf5p, and theEscherichia coli Ψ synthase truB/P35. We therefore tested whether Cbf5p is required for synthesis of Ψ in the yeast rRNA. After genetic depletion of Cbf5p, formation of Ψ in the pre-rRNA is dramatically inhibited, resulting in accumulation of the unmodified rRNA. Protein A-tagged Cbf5p coprecipitates all tested members of the box H + ACA snoRNAs but not box C + D snoRNAs or other RNA species. Genetic depletion of Cbf5p leads to depletion of all box H + ACA snoRNAs. These include snR30, which is required for pre-rRNA processing. Depletion of Cbf5p also results in a pre-rRNA processing defect similar to that seen on depletion of snR30. We conclude that Cbf5p is likely to be the rRNA Ψ synthase and is an integral component of the box H + ACA class of snoRNPs, which function to target the enzyme to its site of action.