Recently, a study reported a primary culture system for tracheal epithelial cells from an embryonic day (E) 17 chick embryo, in which dissociated culture cells exhibited ciliary movement and were positive for the expression of pan-cytokeratin [33] . In addition, the global profile of mRNA expression in the cultured cells showed that cytokeratin 14 (K14), a basal cell marker, was highly expressed. However, the protein expression of cell-type specific markers and the existence of mucin-secreting goblet cell are not well-characterized. In this study, we developed a novel culture system to isolate, amplify and passage chicken tracheal epithelial cells in an efficient manner. The avian tracheal epithelial (ATE) cell types were identified using immunocytostaining, and the ratio of cell types is statistically illustrated. We further demonstrate that primary ATE cells support IBV replication. The susceptible cell types and the effect of glycosaminoglycan (GAG) on IBV attachment to ATE cells was also investigated.