Topoisomerase IIIalpha (topo IIIalpha), a member of the conserved Type IA subfamily of topoisomerases, is required for the cell proliferation in mitotic tissues, but has a lesser effect on DNA endoreplication. The top3alpha gene encodes two forms of protein by utilizing alternative translation initiation sites: one (short form) with the nuclear localization signal only, exclusively localized in the nuclei, and the other (long form), retaining a mitochondrial import sequence at the N-terminus and the nuclear localization sequence at the C-terminus, localized primarily in the mitochondria, though with a small portion in the nuclei. Both forms of topo IIIalpha can rescue the viability of null mutants of top3alpha. No apparent defect is associated with the flies rescued by the long form; short-form-rescued flies (referred to as M1L), however, exhibit defects in fertilities. M1L females are sterile. They can lay eggs but with mitochondrial DNA (mtDNA) copy number and ATP content decreased by 20- and 2- to 3-fold, respectively, and they fail to hatch. Of the newly eclosed M1L males, 33% are completely sterile, whereas the rest have residual fertilities that are quickly lost in 6 days. The fertility loss of M1L males is caused by the disruption of the individualization complex and a progressive loss of germ-line stem cells. This study implicates topo IIIalpha in the maintenance of mtDNA and male germ-line stem cells, and thus is a causative candidate for genetic disorders associated with mtDNA depletion.