We demonstrate that a group I intron-derived ribozyme from the opportunistic pathogen Pneumocystis carinii can bind an RNA in trans and excise from within it an internal segment, resulting in the splicing of the remaining ends of the RNA back together (the trans excision-splicing reaction). The reaction is intramolecular with regard to substrate. The ribozyme targets its substrate by base pairing with two or three noncontiguous regions on the substrate, and the reaction occurs through a nucleotide cofactor independent mechanism. The excised segment can be as long as 28 nucleotides, or more, and as little as one nucleotide. The potential usefulness of this reaction is demonstrated by engineering a ribozyme that excises the triplet-repeat expansion region from a truncated myotonic dystrophy protein kinase transcript mimic in vitro.