In mouse oocytes soon after collection, perivitelline space could be seen in about half of them and its mean size was 0.28 μm. After 6 hrs of in vivo and in vitro maturation, perivitelline space appeared in all oocytes. In oocytes in the process of maturation in vivo the mean size of perivitelline space significantly increased 6 hrs after hCG injection and reached 7.00 μm at 14 hrs after the hCG injection. On the other hand, perivitelline space in cultured oocytes enlarged significantly at 2 hrs culture. Thereafter, perivitelline space in oocytes continued to enlarge with culture time and reached 5.40 μm at 14 hrs of maturation culture. The size of perivitelline space in oocytes 14 hrs after the hCG injection was significantly larger than that in oocytes cultured for 14 hrs. The fertilization rates of oocytes matured in vivo (85%) did not differ from that of oocytes matured in vitro (80%). However, the incidence of polyspermy in oocytes matured in vivo was 4%, which was significantly lower than the 16% of oocytes matured in vitro. These findings suggest there could be a relationship between the size of the perivitelline space and the incidence of polyspermy in mouse oocytes.