The RNA splicing endonuclease is responsible for recognition and excision of nuclear tRNA and all archaeal introns. Despite the conserved RNA cleavage chemistry and a similar enzyme assembly, currently known splicing endonuclease families have limited RNA specificity. Different from previously characterized splicing endonucleases in Archaea, the splicing endonuclease from archaeum Sulfolobus solfataricus was found to contain two different subunits and accept a broader range of substrates. Here, we report a crystal structure of the catalytic subunit of the S.solfataricus endonuclease at 3.1 angstroms resolution. The structure, together with analytical ultracentrifugation analysis, identifies the catalytic subunit as an inactive but stable homodimer, thus suggesting the possibility of two modes of functional assembly for the active enzyme.