The pseudouridylation of ribosomal RNA of Saccharomyces carlsbergensis was investigated with respect to its timing during the maturation of rRNA and its sequence specificity. Analysis of 37-S RNA, the common precursor to 17-S, 5.8-S and 26-S rRNA and most probably the primary ribosomal transcript, shows that this RNA molecule contains already most if not all of the 36-37 pseudouridine residues found in the mature rRNAs. Thus pseudouridylation is, like 2'-0-ribosemethylation, an early event in the maturation of rRNA, taking place immediately after, or even during, transcription. The data presented show that the non-conserved sequences of 37-S precursor rRNA contain very few pseudouridine residues if any. The pseudouridine residues within the rRNA sequences are apparently clustered to a certain degree as can inferred from the occurrence of a single oligonucleotide containing 3 pseudouridines, which was obtained by digestion of 26-S rRNA with ribonuclease T1.