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CD9 is a membrane protein belonging to the tetraspanin family. Despite CD9''s broad tissue distribution, the only abnormality observed in CD9-deficient mice was infertility of females, which was responsible for a defect in the sperm–egg fusion process. However, the function of CD9 in sperm–egg fusion is not clear at all because the technique to analyze the activity of molecules in sperm–egg fusion has not been established. We demonstrated that the exogenous mouse CD9, expressed by polyadenylated mRNA injection at the germinal-vesicle stage oocytes, was precisely localized to the egg plasma membrane, and the expression reversed the infertility of CD9−/− eggs. Then, two other tetraspanins, human CD9 and mouse CD81, overexpressed with this technique on CD9−/− eggs restored the fertilization rate up to ∼90 and ∼50% against that of wild type eggs, respectively. Moreover, in the presence of an anti-mouse CD9 mAb, which blocks sperm–egg fusion, expression of human CD9 or mouse CD81 on eggs also rescued the fusibility. These results suggested that human CD9 plays a crucial role in human fertilization, and mouse CD81 has the potential to compensate for CD9 function in sperm–egg fusion. In addition, the polyadenylated mRNA injection is effective for molecular analysis of sperm–egg fusion. [Copyright &y& Elsevier]