Transgenesis can be achieved in mice by retroviral transduction of male germ line stem cells (GSCs). However, the transduction efficiency by a Moloney murine leukemia virus (MMLV)-based vector is low, probably due to the characteristically slow cell cycle of stem cells. Since lentiviral vectors can transduce non-dividing cells, they have the potential to efficiently transduce GSCs. Here we report that male GSCs of mice can be transduced in vitro by a lentiviral vector and generate complete spermatogenesis when transplanted into infertile host testes. Transduction efficiencies were comparable to those for MMLV transduction using similar experimental conditions. The results suggest that both lentiviral and MMLV vectors could be effective in transducing GSCs of other species. In addition, these and previous studies suggest that transduction of immature donor stem cells transplanted into immature recipient testes will provide the most efficient system for male germ line modification.