We developed a method of culturing bursal epithelial cells under serum-free condition. Bursal cells sampled from 14 days of embryogenesis were cultured in DMEM/ Ham's F12 1 : 1 mixture medium supplemented with epidermal growth factor, insulin, transferrin, sodium selenite, hydrocortisone and 2-aminoethanol. A monolayer, in which polygonal cells were extensively observed, entirely covered the well after 2 to 3 weeks of incubation. The cultured cells were maintained for 5 months without any apparent morphological abnormalities. Polygonal cells in wells cultured for 3 weeks or 5 months were reacted with anti-keratin antibody, indicating these cells are cytokeratin-positive cells. These results suggest that most cells proliferated under a serum-free condition are epithelial cells. It seems possible to precisely analyze presumable factors in the supernatant of bursal epithelial cell culture. © 2003, Japan Poultry Science Association. All rights reserved.