RNA splicing in archaea requires at least an endonuclease and a ligase, as is the case for the splicing of eukaryal nuclear tRNAs. Splicing endonucleases from archaea and eukarya are homologous, although they differ in subunit composition and substrate recognition properties. However, they all produce 2′,3′ cyclic phosphate and 5′-hydroxyl termini. An in vitro-transcribed, partial intron-deleted Haloferax volcanii elongator tRNAMet has been used to study splicing by H. volcanii cell extracts. Substrates and products were analyzed by nearest neighbor analyses using nuclease P1 and RNase T2, and fingerprinting analyses using acid-urea gels in the first dimension and gradient thin layer chromatography in the second dimension. The results suggest that 2′,3′ cyclic phosphate at the 3′ end of the 5′ exon is converted into the splice junction phosphate forming a 3′,5′-phosphodiester linkage. This resembles the animal cell type systems where the junction phosphate preexists in the transcript, and differs from yeast type systems, where GTP is the source of junction phosphate.