Assisted reproductive techniques involving isolation, culture, and transplantation of spermatogonial stem cells (SSCs) have the potential to create transgenic livestock and to treat male infertility caused by cancer treatments such as chemotherapy or radiation. Because stem cells may need to be preserved for several years before reintroduction to the patients' testes, efficient SSC cryopreservation techniques need to be developed. SSCs can reinitiate spermatogenesis in recipient testes after freezing; however, optimal cryopreservation protocols have not been identified. The objective of this study was to develop an efficient cryopreservation method for SSCs using permeable cryoprotectant agents (PCAs) or additive cryoprotectant agents (ACAs). To identify an efficient cryopreservation method, populations of mouse testis cells enriched for SSCs were cultured in vitro and frozen using conventional freezing media containing various PCAs or ACAs for 1 wk or 1, 3, 6, 12, or 24 mo. Additionally, various molecular weights and concentrations of polyethylene glycol (PEG) were evaluated. Recovery rate, culture potential, and stem cell activity were significantly greater for cells frozen in 2.5% PEG with a molecular weight of 1000 compared to other treatment groups. These cells also retained the ability to colonize recipient testes, generate normal spermatogenesis, and contribute to viable offspring. The systematic analysis of many cryoprotectant agents indicates that 2.5% PEG (molecular weight 1000) is the most effective agent for efficient SSC cryopreservation.