In the mitochondria of trypanosomatid protozoa the precursors of messenger RNAs (pre-mRNAs) have their coding information remodeled by the site-specific insertion and deletion of uridylate (U) residues. Small trans-acting guide RNAs (gRNAs) supply the genetic information for this RNA editing. An in vitro system was developed to study the mechanism of U insertion into pre-mRNA. U-insertion editing occurs through a series of enzymatic steps that begin with gRNA-directed pre-mRNA cleavage. Inserted U’s are derived from free uridine triphosphate and are added to the 3’ terminus of a 5’ pre-mRNA cleavage product. gRNA specifies edited RNA sequence at the subsequent ligation step by base pairing-mediated juxtaposition of the 3’ cleavage product and the processed 5’ cleavage product. gRNA/pre-mRNA chimeras, purported intermediates, seem to be abortive end products of the same reaction.