This chapter describes the preparation of respiratory tract tissue from both mice and rats for the isolation of respiratory tract dendritic cells (RTDC). The methods describe in detail the preparation of cells from the respiratory tract tissue of the main conducting airways (representing mucosal populations) and peripheral lung (representing predominantly interstitial populations) in both rodent species. Our research in this area has found that these anatomical sites differ in their composition of antigen-presenting cell (APC) types including RTDC, and that phenotypic and functional differences exist in RTDC isolated from these sites. We predominantly use a flow cytometry-based approach to identify and sort RTDC as this is the most accurate way of isolating RTDC subsets in an environment where many typical dendritic cell surface markers are shared by other APC populations.