To generate functional tRNA molecules, precursor RNAs must undergo several processing steps. While the enzyme that generates the mature tRNA 5'-end, RNase P, has been thoroughly investigated, the 3'-processing activity is, despite its importance, less understood. While nothing is known about tRNA 3'-processing in archaea, the phenomenon has been analysed in detail in bacteria and is known to be a multistep process involving several enzymes, including both exo- and endonucleases. tRNA 3'-end processing in the eukaryotic nucleus seems to be either exonucleolytic or endonucleolytic, depending on the organism analysed, whereas in organelles, 3'-end maturation occurs via a single endonucleolytic cut. An interesting feature of organellar tRNA 3'-processing is the occurrence of overlapping tRNA genes in metazoan mitochondria, which presents a unique challenge for the mitochondrial tRNA maturation enzymes, since it requires not only the removal but also the addition of nucleotides by an editing reaction.