The oolemma of the second metaphase mouse oocyte has a relatively large domain in the peri-polar body region that is non-permissive for sperm attachment. In this study, certain biochemical components of this non-permissive domain were examined and compared with permissive regions to investigate the molecular basis of an oolemma that becomes functionally polarized during fertilization; an attempt was also made to determine whether similarities exist in the formation of abnormally occurring non-permissive domains in human oocytes. In the present study, microdomains composed of lipid rafts enriched in the ganglioside GM1 provided a platform for sperm docking at the initial stage of fertilization and their disruption led to sperm attachment defects resembling those associated with fertilization failure in human IVF. The regulation of GM1 microdomain density is suggested to involve the oolemmal protein annexin 2 and the magnitude of the inner mitochondrial membrane potential (ΔΨm) in the adjacent subplasmalemmal cytoplasm. These studies also showed that a rapid focal reduction in GM1, f-actin, annexin 2 and cholesterol occurs at the site of sperm docking and attachment whereas the distribution of fertilization-associated proteins CD9 and FOLR4 remains unchanged, suggesting that they may not participate in the earliest phases of fertilization.