tRNA 3' processing endoribonuclease (3' tRNase) is an enzyme responsible for the removal of a 3' trailer from precursor tRNA (pre-tRNA). We purified approximately 85 kDa 3' tRNase from pig liver and determined its partial sequences. BLAST search of them suggested that the enzyme was the product of a candidate human prostate cancer susceptibility gene, ELAC2, the biological function of which was totally unknown. We cloned a human ELAC2 cDNA and expressed the ELAC2 protein in Escherichia coli. The recombinant ELAC2 was able to cleave human pre-tRNA(Arg) efficiently. The 3' tRNase activity of the yeast ortholog YKR079C was also observed. The C-terminal half of human ELAC2 was able to remove a 3' trailer from pre-tRNA(Arg), while the N-terminal half failed to do so. In the human genome exists a gene, ELAC1, which seems to correspond to the C-terminal half of 3' tRNase from ELAC2. We showed that human ELAC1 also has 3'-tRNase activity. Furthermore, we examined eight ELAC2 variants that seem to be associated with the occurrence of prostate cancer for 3'-tRNase activity. Seven ELAC2 variants which contain one to three amino acid substitutions showed efficient 3'-tRNase activities, while one truncated variant, which lacked a C-terminal half region, had no activity.