Accurate tRNA processing is crucial for human mitochondrial genome expression, but the mechanisms of mt-tRNA cleavage and the key enzymes involved in this process are poorly characterized. At least two activities are required for proper mt-tRNA maturation: RNase P cleaving precursor molecules at the 5' end and tRNase Z at the 3' end. In human mitochondria only RNase P has been identified so far. Using RT-PCR and northern blot analyses we found that silencing of the human ELAC2 gene results in impaired 3' end of mt-tRNAs. We demonstrate this for several mitochondrial tRNAs, encoded on both mtDNA strands, including tRNA (Val) , tRNA (Lys) , tRNA (Arg) , tRNA (Gly) , tRNA (Leu(UUR)) and tRNA (Glu) . The silencing of the MRPP1 gene that encodes a subunit of mtRNase P resulted in inhibition of both 5' and 3' processing. We also demonstrate the double mitochondrial/nuclear localization of the ELAC2 protein using immunofluorescence. Our results indicate that ELAC2 functions as a tRNase Z in human mitochondria and suggest that mt-tRNase Z preferentially cleaves molecules already processed by the proteinaceous mtRNase P.