A major route of infection of avian influenza virus (AIV) and Newcastle disease virus (NDV) in chickens is through cells of the respiratory epithelium. Here we describe the development of a method for culture of tracheal epithelial cells from chicken embryos as well as their use in studies of infection with avian respiratory viruses such as low-pathogenicity AIV and lentogenic NDV. Positive immunostaining for cytokeratin, the presence of cilia and microvilli, and microarray analysis of transcribed RNA demonstrated that the isolated cells were epithelial in nature. Infection of the epithelial cell cultures with AIV and NDV was demonstrated using immunofluorescence or green fluorescence protein fluorescence microscopy, respectively. Growth curves of AIV and NDV in tracheal epithelial cells revealed that tracheal epithelial cells can fully support AIV and NDV growth and reinfection. This system, which mimics that of the natural infection, will be useful to study the mechanisms of early viral infection and cellular host transcriptional responses.