The freshly ovulated ovum in avian species is surrounded by the inner perivitelline layer (IPVL). For successful fertilization, sperm must attach and penetrate the IPVL. In the domestic chicken six distinct zona pellucida genes have been identified (ZPA, ZPB1, ZPB2, ZPC, ZPD and ZPX1). ZPB1 is produced by the liver and transported to developing follicles, while ZPA, ZPB2, ZPC, ZPD and ZPX1 are synthesized by follicular granulosa cells. In turkey hens, differences in mRNA expression of the ZP proteins are associated with preferential binding of sperm at the germinal disc (GD) region and with differences in fertility among genetic strains of hens.The expression of ZPB2 mRNA was investigated by real time RT-PCR in theca and granulosa cells from the developing preovulatory follicles of broiler breeder hens. Expression of ZPB2 mRNA was highest in granulosa cells isolated from follicles with a diameter less than 2 mm, followed by 2-5 mm diameter and F4 follicles. Expression of ZPB2 mRNA in the F2 and F3 follicle was lower than the F4 follicle. ZPB2 mRNA was not detected in the granulosa cells isolated from 5-8 mm diameter, or 8-12 mm diameter follicles or in theca samples from hierarchical follicles. Theca cell expression of ZPB2 mRNA was confined to the theca cells from the smallest follicles.The mRNA expression of ZPB1, ZPB2 and ZPC was analyzed in four broiler breeder hen genetic lines designated as B, G, O and R. Expression of ZPB1, ZPB2, and ZPC mRNA was detected in all genetic lines. Expression of ZPB2 mRNA differed between NGD and GD granulosa cells. Hepatic ZPB1 mRNA levels were lower in line O than line B. In genetic strains B, G, and R, ZPC mRNA expression was higher in NGD than GD granulosa cells. However, in genetic strain O the expression of ZPC mRNA did not differ between NGD and GD granulosa cells.Antibodies against chicken ZPB1, ZPB2 and ZPC were produced and validated for subsequent use in determining if differences in the mRNA expression of the ZP proteins are also present at the protein expression level.