Editing of mitochondrial mRNAs in kinetoplastid protozoa occurs by a series of enzymatic steps that insert and delete uridylates (U's) as specified by guide RNAs (gRNAs). The characteristics of the 3' exonuclease activity that removes the U's following cleavage during deletion editing were determined by using an in vitro precleaved deletion assay that is based on ATPase subunit 6 pre-mRNA and gA6[14] gRNA. The exonuclease in partially purified editing complexes is specific for U's. The specificity occurs in the absence of gRNA, but its activity is enhanced by the presence of gRNA. The 3' pre-mRNA fragment enhances the specificity, but not the efficiency, of U removal. The activity is sensitive to the 5' phosphate of the 3' fragment, which is not required for U removal. The ability of the 3' U's to base pair with purines in the gRNA protects them from removal, suggesting that the U-specific 3' exonuclease (exoUase) is specific for U's which are not base paired. ExoUase is stereospecific and cannot remove (Rp)alpha-thio-U. The specificity of the exoUase activity thus contributes to the precision of RNA editing.