Investigating intestinal physiology in vitro remains challenging due to the lack of an effective primary enterocyte culture system. Recently developed protocols for growing organoids containing crypts and villus from adult mouse intestinal epithelium in Matrigel present an attractive alternative to the classical techniques. However, these approaches require the use of sophisticated and expensive serum-free medium supplemented with epithelial growth factor (EGF), Wnt agonist (R-spondin 1), and bone morphogenetic protein inhibitor (Noggin) in high concentrations. Here we demonstrate that is possible to use an isolated chicken embryonic intestinal epithelium to create such an organoid culture. Structures formed in Matrigel matrix in the first two days following isolation survive and enlarge during ensuing weeks. They have the appearance of empty spheres and comprise cells expressing cytokeratin (an epithelial cell marker), villin (a marker of enterocytes), and Sox-9 (a transcription factor characteristic of progenitors and stem cells of intestinal crypts). With chicken embryonic tissue as a source of organoids, prostaglandin E2 is as effective as R-spondin 1 and Noggin in promoting sustained growth and survival of epithelial spheroids.