The structure of the zona pellucida (ZP) was analyzed in mouse oocytes collected soon after ovulation and in others retrieved 20 h after. The conventional methods for electron microscopy and the alcoholic PTA staining procedure which preferentially contrasts lysine-rich proteins were employed. The ZP of aged oocytes showed several structural changes which were particularly observed after using the PTA procedure. In 82.14% of the aged oocytes the ZP appeared clearly composed of two different regions: an inner dense and an outer of low density. The ZP showed a fibrillar banded structure with a parallel arrangement of fibrillar threads in both the outer and inner regions. The in vitro fertilization analysis showed that only 16.85% of the aged gametes attained the two cell embryo stage in comparision to 66.93% shown by the freshly ovulated eggs. The non-fertilized oocytes showed that no sperm penetration through the ZP occurred.