RNA editing corrects a 4C-A69 mismatch to a conventional 4T-A69 Watson-Crick base pair in the acceptor stem of the mitochondrially encoded tRNAPhe in plants. In vitro processing of edited and unedited Oenothera tRNA Phe precursor RNAs with pea mitochondrial protein extracts shows a significant effect of this RNA-editing event on the efficiency of 5' and 3' processing. While mature tRNA molecules are rapidly generated by in vitro processing from edited precursors, the formation of mature tRNAs from unedited pre-tRNAs is considerably reduced. Primer extension analyses of in vitro processing products show that processing at both 5' and 3' termini is governed by the RNA-editing event. Investigation of edited and unedited precursor RNAs by lead cleavage experiments reveals differences in the higher order structures of the pre-tRNAs. The differing conformations are most likely responsible for the altered processing efficiencies of edited and unedited precursor molecules. RNA editing of the tRNAPhe precursors is thus a prerequisite for efficient excision of the mature tRNAPhe in vitro. Hence RNA editing might be involved in regulating the amount of mature tRNAPhe in the steady state RNA pool of mitochondria in higher plants.