Recipient preparation is of prime importance for the successful transplantation of spermatogonial stem cells (SSCs). Busulfan destroys endogenous germs cells and is commonly used for recipient preparation. However, busulfan produces significant side effects, including systemic toxicity, and it is lethal in certain species. The side effects associated with busulfan compromise the efficiency of SSC transplantation and threaten the safety of recipients. Here, we show that heat shock treatment of testes can be used as an alternative to busulfan treatment. Fourteen days after heat shock treatment, mice received a testicular injection of donor germ cells expressing enhanced green fluorescent protein (EGFP). Busulfan-treated mice were used as controls. Two months after transplantation, the number (12 ± 1 mm) and length (30.46 ± 5.23 mm) of EGFP-expressing testicular colonies in heat shock-treated recipients were not significantly different from those in busulfan-treated recipients. Furthermore, healthy EGFP-expressing offspring were obtained after intracytoplasmic injection of round spermatids recovered from heat shock-treated recipients. This result indicates that donor SSCs undergo complete spermatogenesis in the heat shock-treated testes of recipients. Our findings demonstrate the feasibility of using heat shock for the preparation of recipients before SSC transplantation in mice. Heat shock may prove to be useful for recipient preparation in mammalian species in which busulfan produces significant toxicity.