Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ 2603 in Pyrococcus abyssi 23S rRNA and of Ψ 55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ 55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ 2607 (Ψ 2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA-and Gar1-dependent activity of Cbf5. Pseudouridine (Ψ) is the most abundant RNA modification (for review 1-3). This modified nucleoside is generated by site-specific C5-ribosyl isomerization of uridine (U) in pre-existing RNA transcripts. Formation of Ψ in RNAs can increase base stacking 4 and rigidify the sugar-phosphate backbone 1,5,6. More specifically, Ψ can stabilize RNA 3D conformation by water bridge formation, as demonstrated in tRNAs 7,8. A cluster of Ψs can modulate the conformation of a large RNA domain as shown for helix 69-H69-in rRNA 9. It is now firmly established that some evolutionarily conserved Ψs in tRNAs and rRNAs are required for accurate function of these non-coding RNAs (ncRNAs) in translation (for review 2 , and 10-20). Similarly, some of the Ψs in U-rich small nuclear RNAs (UsnRNAs) are important for pre-mRNA splicing 2,21,22. Moreover, recent transcriptome-wide maps of pseudou-ridylation detected a large number of Ψs in eukaryotic mRNAs 23,24 but their precise function is not yet firmly established. The conversion of uridine (U) into Ψ is catalyzed by specific RNA:Ψ-synthases (PUS) (for review 25). Five families of PUS have been defined based on protein sequence conservation with each of the bacterial TruA, TruB, TruD, RluA, and RsuA proteins (for review 25). Comparative analysis of eukaryotic genomes revealed the presence of members of the same five families of PUS in Eukarya. Studies on archaea identified a rather limited