The specificity of reactions catalyzed by the Tetrahymena pre-rRNA intervening sequence (1%) was studied using site-specific mutagenesis. Two sequences required for Ysplice-site selection during selfsplicing were defined. Single-base changes in either a 5’exon sequence or a 5’exon-binding site within the IVS disrupt their abillty to pair and result in inefficient or inaccurate splicing. Combinations that restore complementarity suppress the effect of the singlebase changes. Sequence alterations in the 5’exonbinding site also change the specificity of two other reactions: intermolecular exon ligation (frans-splicing) and the enzymatic nucleotldyltransferase activity of the IVS RNA. Thus the substrate specificity of an RNA enzyme can be changed in a manner predictable by the rules of Watson-Crick base-pairing.