The aim of this study was to identify the avian β-defensin-12 (AvβD-12) protein in the ovarian follicles and to determine the changes in its localization during follicular growth and in response to lipopolysaccharide (LPS). Anti-AvβD-12 polyclonal antibody was raised using synthetic peptide. White Leghorn laying hens were i.v. injected with or without LPS (1mg/kg BW). White follicles and yellow follicles (the largest and the third largest follicles; F₁ and F₃, respectively) were collected before and after 12 or 24h of LPS injection (n=3 in each group). Immunocytochemistry using an avidin-biotin-peroxidase complex method was performed using their paraffin sections. The immunoreactive AvβD-12 (irAvβD-12) was not found in the white follicles. The irAvβD-12 was localized in the capillary-associated cells in the outer theca interna, granulosa cells and perivitelline layer of yellow follicles. The irAvβD-12 in the theca interna and granulosa layers of yellow follicles was decreased by 12h and disappeared by 24h of LPS injection. Perivitelline layer was kept positive for irAvβD-12 even after 24h of LPS injection. No significant difference was found in the distribution of irAvβD-12 between F₃ and F₁ for the above results. No immunoreaction products were observed in the sections incubated with absorbed antibody of AvβD-12 instead of first antibody (control staining). These results suggest that the theca interna, granulosa and perivitelline layers contain AvβD-12. Its amount is likely increased with follicular growth from a white follicle to a yellow follicle, whereas the protein may be released from the cells by LPS stimulation, probably as a host defense response.