RNA editing in Trypanosoma brucei produces mature mRNAs by posttranscriptional insertion and deletion of uridylates (Us) by a series of catalytic steps, which include endoribonucleolytic cleavage, 3' terminal addition or removal of Us, and RNA ligation. Preedited mRNA (pre-mRNA) and guide RNA (gRNA) that are mutated at or near the editing site (ES) were used to examine the effects on the specificity of in vitro editing. Sequences that are not predicted to form a gRNA/pre-mRNA base pair immediately 5' to the ES still supported accurate editing. Substitution of a non-U nucleotide at various positions within a stretch of Us that are normally removed from the ES resulted in deletion of only the Us that were 3' to the substituted nucleotide. Overall, ES selection by the endoribonuclease, the specificity of the 3' exoribonuclease for Us, and ligation appear to act in concert to ensure the production of accurately edited RNA.