The relationship between enzyme architecture and substrate specificity among archaeal pre-tRNA splicing endonucleases has been investigated more deeply, by using biochemical assays and model building. The enzyme from Archeoglobus fulgidus (AF) is particularly interesting: it cleaves the bulge-helix-bulge target without requiring the mature tRNA domain, but, when the target is a bulge-helix-loop, the mature domain is required. A model of AF based on its electrostatic potential shows three polar patches interacting with the pre-tRNA substrate. A simple deletion mutant of the AF endonuclease lacking two of the three polar patches no longer cleaves the bulge-helix-loop substrate with or without the mature domain. This single deletion shows a possible path for the evolution of eukaryal splicing endonucleases from the archaeal enzyme.