A sperm factor(s) for oocyte activation during fertilization has not been clearly identified. In this study to elucidate an oocyte activation factor(s), mouse sperm were sonicated and ultra-filtered with a 30 kilo-daltons (KD) cutoff membrane and the ultra-filtrate was then sequentially fractionated over Suporose 12 column and Superdex column, The recovered fractions were micro-injected into Mⅱmouse oocytes and second polar body formation (PBF) was examined. Suporose fraction RV2.10 prepared from sperm extract significantly increased PBF. Of Superdex fractions re-separated from Suporose fraction RV2.10, fraction RV2.12 also had the strongest PBF activity. By analyzing with micro-reverse phase column (URPC), the Superdex fraction RV2.12 appeared to be glutamic acid. In microinjection test, glutamic acid significantly increased PBF. This study suggests that glutamic acid should be a type of sperm factor for second polar body formation related to oocyte activation.