A reproducible and original method for the preparation of chicken intestine epithelial cells from 18-day-old embryos for long-term culture was obtained by using a mechanical isolation procedure, as opposed to previous isolation methods using relatively high concentrations of trypsin, collagenase, or EDTA. Chicken intestine epithelial cells typically expressed keratin and chicken E-cadherin, in contrast to chicken embryo fibroblasts, and they increased cell surface MHC II after activation with crude IFN-γ containing supernatants, obtained from chicken spleen cells stimulated with concanavalin A or transformed by reticuloendotheliosis virus. Eimeria tenella was shown to be able to develop until the schizont stage after 46 hr of culture in these chicken intestinal epithelial cells, but it was not able to develop further. However, activation with IFN-γ containing supernatants resulted in strong inhibition of parasite replication, as shown by incorporation of [³H]uracil. Thus, chicken enterocytes, which are the specific target of Eimeria development in vivo, could be considered as potential local effector cells involved in the protective response against this parasite.