Overlapping tRNA genes in mitochondria of many metazoans introduce a problem for the processing of such polycistronic primary transcripts. Using runoff transcripts and an S100 extract from HeLa cell mitochondria, the processing of the human mitochondrial tRNATyr/tRNACys precursor (carrying an overlap of one base) was investigated: tRNACys is released in its complete form carrying the overlapping residue at the first position, whereas tRNATyr lacks that nucleotide at the discriminator position. Partial deletion of tRNACys or complete replacement by a non-tRNA-like sequence does not alter the processing reaction and indicates that the upstream tRNATyr alone is recognized by a 3'-endonuclease activity. The truncated 3'-end of this tRNATyr is then completed in an editing reaction that incorporates the missing residue. The processing of this tRNA overlap seems to be species-specific, because an overlapping tRNA precursor (tRNASer(AGY)/tRNALeu(CUN)) from opossum mitochondria is not recognized by the human extract. Because processing activities for overlapping and nonoverlapping tRNA precursors could not be separated, it seems that one general activity is responsible for the 3'-end processing of mitochondrial tRNAs and that this activity coevolved with the particular overlap between tRNATyr and tRNACys in human mitochondria, being unable to recognize overlaps between other tRNAs.