Spermatozoa from a number of species can be cryopreserved and then subsequently used to fertilize eggs. However, this technique has several limitations. First, the freezing protocol varies for each species and must be determined empirically, and for some species appropriate methods have not yet been identified. Second, because these cells are fully differentiated, they will not undergo replication when thawed, and recombination of genetic information cannot occur. We now demonstrate, by using the recently developed spermatogonial transplantation technique, that male germline stem cells can be successfully cryopreserved. Donor testis cells isolated from prepubertal or adult mice and frozen from 4 to 156 days at -196 degrees C were able to generate spermatogenesis in recipient seminiferous tubules. Relatively standard preservation techniques were used, suggesting that male germ cells from other species can also be stored for long periods. Because transplanted testis stem cells will ultimately undergo replication and meiotic recombination during spermatogenesis, one might consider these preserved male germ lines as biologically immortal.