tRNase Z is an essential endonuclease responsible for tRNA 3'-end maturation. tRNase Z exists in a short form (tRNase Z(S)) and a long form (tRNase Z(L)). Prokaryotes have only tRNase Z(S), whereas eukaryotes can have both forms of tRNase Z. Most eukaryotes characterized thus far, including Saccharomyces cerevisiae, Caenorhabditis elegans, Drosophila melanogaster, and humans, contain only one tRNase Z(L) gene encoding both nuclear and mitochondrial forms of tRNase Z(L). In contrast, Schizosaccharomyces pombe contains two essential tRNase Z(L) genes (trz1 and trz2) encoding two tRNase Z(L) proteins, which are targeted to the nucleus and mitochondria, respectively. Trz1 protein levels are notably higher than Trz2 protein levels. Here, using temperature-sensitive mutants of trz1 and trz2, we provide in vivo evidence that trz1 and trz2 are involved in nuclear and mitochondrial tRNA 3'-end processing, respectively. In addition, trz2 is also involved in generation of the 5'-ends of other mitochondrial RNAs, whose 5'-ends coincide with the 3'-end of tRNA. Thus, our results provide a rare example showing partitioning of the nuclear and mitochondrial tRNase Z(L) activities between two different proteins in S. pombe. The evolution of two tRNase Z(L) genes and their differential expression in fission yeast may avoid toxic off-target effects. 