Mouse zona pellucida (ZP) proteins are synthesized in developing oocytes and assembled into ZP after their secretion. This study has investigated whether anti-ZP3 antibodies affect ZP assembly. Peptides CP2 and CP3 were used to elicit antibodies to two ZP3 B cell epitopes, ZP3 (335-342) and ZP3 (171-180). Ovulated eggs from mice immunized with a mixture of CP2/CP3 showed an abnormal ZP; importantly, the ZP completely dissolved both in vitro and in vivo 12h after ovulation. Although CP3 immunization resulted also in abnormal ZP, the ZP did not dissociate. Binding of antibodies to the ZP prior to oocyte maturation was requisite, as in vitro incubation of ovulated eggs in combination with the two antibodies failed to induce ZP dissolution. Electron microscopic observation further demonstrated a significant abnormality in ZP structure in CP2/CP3-immunized mice, especially in mature follicles, suggesting that B cell epitopes may be involved in ZP assembly. Though antibody elicited by CP2 has been shown to inhibit fertilization, we now show that antibody induced by CP3 had no effect on fertility. However, immunization with CP3/CP2 resulted in a significantly lower fertility rate than CP2 alone. This suggests that infertility in these mice may be due to an unstable ZP structure. Our model provides a useful tool to study ZP assembly and its structure beyond molecular biology method.