Objective To explore the optimal time for microinjection into fertilized eggs. Methods After superovulation,fertilized eggs of mice at different stages were collected.The male pronuclei of fertilized eggs was observed and calculated at different stages.The optimal time for the occurrence of male pronuclei of fertilized eggs was recorded. Results After injecting human chorionic gonadotropin(HCG) for 23-25 h,the male pronuclei were found in more than 75% fertilized eggs.The female and male pronuclei were clearly visible,and the male pronuclei of fertilized eggs were larger than those at the other stages. Conclusion It is the most suitable time for microinjection into fertilized eggs after HCG injection for 23-25 h.