Accuracy in transfer RNA (tRNA) splicing is essential for the formation of functional tRNAs, and hence for gene expression, in both Eukaryotes and Archaea. The specificity for recognition of the tRNA precursor (pre-tRNA) resides in the endonuclease, which removes the intron by making two independent endonucleolytic cleavages. Although the eukaryal and archaeal enzymes appear to use different features of pre-tRNAs to determine the sites of cleavage, analysis of hybrid pre-tRNA substrates containing eukaryal and archaeal sequences, described here, reveals that the eukaryal enzyme retains the ability to use the archaeal recognition signals. This result indicates that there may be a common ancestral mechanism for recognition of pre-tRNA by proteins.