Pseudouridine (psi), the most common single modified nucleoside in ribosomal RNA, has been positioned in the small subunit (SSU) and large subunit (LSU) RNAs of a number of representative species. Most of the information has been obtained by application of a rapid primed reverse transcriptase sequencing technique. The locations of these psi residues have been compared. Many sites for psi are the same among species, but others are distinct. In general, the percentage psi in multicellular eukaryotes is greater than in prokaryotes. In LSU RNA, the psi residues are strongly clustered in three domains, all of which are near or connected to the peptidyl transferase center. There is no apparent clustering of psi in SSU RNA. The psi sites in LSU RNA overlap those for the methylated nucleosides, but this is not the case in SSU RNA. There are 265 psi sites known to nucleotide resolution, of which 246 are in defined secondary structures, and 112 of these are in nonidentical structural contexts. All 246 psi sites can be classified into five structural types. Two Escherichia coli psi synthases have been cloned and characterized, one for psi 516 in SSU RNA and one for psi 746 in LSU RNA. The psi 746 synthase recognizes free RNA, but the psi 516 enzyme requires an intermediate RNP particle. Possible functional roles for psi in the ribosome are discussed.