Genes belonging to the piwi family are required for stem cell self-renewal in diverse organisms. We cloned mouse homologues of piwi by RT-PCR using degenerative primers. The deduced amino acid sequences of mouse homologues MIWI and MILI showed that each contains a well-conserved C-terminal PIWI domain and that each shares significant homology with PIWI and their human counterparts HIWI. Both miwi and mili were found in germ cells of adult testis by in situ hybridization, suggesting that these genes may function in spermatogenesis. Furthermore, mili was expressed in primordial germ cells (PGCs) of developing mouse embryos and may therefore play a role during germ cell formation. MIWI may be involved in RNA processing or translational regulation, since MIWI was found to possess RNA binding activity. Our data suggest that miwi and mili regulate spermatogenesis and primordial germ cell production.