The complex process of spermatogenesis is regulated by various factors. In the present study, the in vitro effects of epidermal growth factor (EGF), follicle stimulating hormone (FSH) and testosterone on spermatogonial cell colony formation were investigated, and the best colonising factor was chosen for treating cells before transplantation. Sertoli and spermatogonial cells were isolated from neonatal mouse testes. The identity of the cells was confirmed through analysis of morphology, alkaline phosphatase activity, immunoreactivity and transplantation. Co-cultured Sertoli and spermatogonial cells were treated with EGF, FSH and testosterone before colony assay. Results indicated that EGF is the best factor for in vitro colonisation of spermatogonial cells, but transplantation of the EGF-treated group did not show any significant change compared with the control groups. In conclusion, EGF increased in vitro colonisation of spermatogonial cells, but, as a result of differential effects, did not influence transplantation efficiency.