An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation.Busulfan has been commonly used to develop such a model,but 30％-87％ of mice die when administered an intraperitoneal injection of 40 mg kg-1.In the present study,hematoxylin and eosin staining,Western blot,immunofluorescence,and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20,30,and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0,9,18,27,36,and 63 days).The survival rate of the mice was 100％.When the mice were treated with 40 mg kg-1 busulfan,dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36.In addition,the gene expressions of glial cell line-derived neurotrophic factor (GDNF),fibroblast growth factor 2 (FGF2),chemokine (C-X-C Motif) ligand 12 (CXCL12),and colony-stimulating factor 1 (CSF1) were moderately increased by day 36.A 63-day,long-term observation showed the rare restoration of endogenous germ cells in the testes,suggesting that the potential period for SSC transplantation was between day 36 and day 63.Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.