The single 23S rRNA gene of the archaeon Staphylothermus marinus exhibits two introns which, at the RNA level, are located in highly conserved regions of domains IV and V. The RNA introns, which are 56 and 54 nucleotides long, respectively, can form single hairpin structures. In vivo, RNA splicing occurs efficiently, whereas in vitro pre-rRNA transcripts containing each intron were cleaved efficiently when incubated with archaeal cell extracts but were poorly ligated. The introns are cleaved by a mechanism which differs from the mechanisms of eukaryotic rRNA introns but resembles those of the rRNA intron of Desulfurococcus mobilis and the archaeal tRNA introns. The cleavage enzyme recognizes and cuts a putative bulge-helix-bulge structure that can form at the archaeal exon-intron junctions. Using a phylogenetic sequence comparison approach, we define the parts of this structural feature that are essential for cleavage. We also provide evidence for conformational changes occurring in the S. marinus 23S RNA, after cleavage, at both exon-exon junctions, which may account for the low yields of ligation observed in vitro.