Three enterocyte cell clones were established in vitro from the intestine of a PA12 hen embryo. These cells exhibited epithelioid morphology and grew as monolayers. The cells were continuously propagated in culture up to 250 passages. Gradual increase in growth rate with time and in anchorage-independent growth in both agar and agarose showed that the three cell clones spontaneously transformed in vitro. The clones were heteroploid with one marker chromosome. Interestingly, they had features of partly differentiated enterocytes, especially microvilli, junctions connecting adjacent cells (tight junctions, desmosomes, hemidesmosomes, gap junctions), villin and cytokeratins. In addition, cells expressed brush border enzyme activity and transepithelial resistance. The fact that the levels of dipeptidyl peptidase IV (DPP-IV) and alkaline phosphatase activities fluctuated according to culture time and that MHC class II was induced by activation of cells with interferon suggested that the state of differentiation of the 3 cell clones could be modified in vitro. These clones are the first established avian enterocyte cell clones to be described. Because each cell clone exhibited differences in the level of differentiation and sensitivity to Salmonella infection, their use will allow comparative investigations concerning markers of differentiation of avian enterocytes and infection by host-adapted bacteria and parasites.