Although tRNase Z from various organisms was shown to process nuclear tRNA 3' ends in vitro, only a very limited number of studies have reported its in vivo biological functions. tRNase Z is present in a short form, tRNase Z(S), and a long form, tRNase Z(L). Unlike Saccharomyces cerevisiae, which contains one tRNase Z(L) gene (scTRZ1) and humans, which contain one tRNase Z(L) encoded by the prostate-cancer susceptibility gene ELAC2 and one tRNase Z(S), Schizosaccharomyces pombe contains two tRNase Z(L) genes, designated sptrz1(+) and sptrz2(+). We report that both sptrz1(+) and sptrz2(+) are essential for growth. Moreover, sptrz1(+) is required for cell viability in the absence of Sla1p, which is thought to be required for endonuclease-mediated maturation of pre-tRNA 3' ends in yeast. Both scTRZ1 and ELAC2 can complement a temperature-sensitive allele of sptrz1(+), sptrz1-1, but not the sptrz1 null mutant, indicating that despite exhibiting species specificity, tRNase Z(L)s are functionally conserved among S. cerevisiae, S. pombe and humans. Overexpression of sptrz1(+), scTRZ1 and ELAC2 can increase suppression of the UGA nonsense mutation ade6-704 through facilitating 3' end processing of the defective suppressor tRNA that mediates suppression. Our findings reveal that 3' end processing is a limiting step for defective tRNA maturation and demonstrate that overexpression of sptrz1(+), scTRZ1 and ELAC2 can promote defective tRNA 3' processing in vivo. Our results also support the notion that yeast tRNase Z(L) is absolutely required for 3' end processing of at least a few pre-tRNAs even in the absence of Sla1p.