Introns in protein-coding genes are ubiquitous in eukaryotic cells, but pre-mRNA splicing has yet to be reported in archaeal and its viral genomes. We present evidence of introns in genes encoding a homolog of eukaryotic Cbf5p (centromere-binding factor 5; a subunit of a small nucleolar ribonucleoprotein) in three Archaea; Aeropyrum pernix, Sulfolobus solfataricus and Sulfolobus tokodaii. Splicing of pre-mRNAs in vivo was demonstrated by reverse transcriptase-mediated polymerase chain reaction. The exon-intron boundaries of these genes are predicted to be folded into a structure similar to the bulge-helix-bulge motif, suggesting that splicing of these pre-mRNAs probably depends on the splicing system elucidated for archaeal pre-tRNAs and rRNAs.