Antigen transport from the airway mucosa to the thoracic lymph nodes (TLNs) was studied in vivo by intratracheal instillation of fluorescein isothiocyanate (FITC)-conjugated macromolecules. After instillation, FITC⁺ cells with stellate morphology were found deep in the TLN T cell area. Using flow cytometry, an FITC signal was exclusively detected in CD11c^med-hi/major histocompatibility complex class II (MHCII)^hi cells, representing migratory airway-derived lymph node dendritic cells (AW-LNDCs). No FITC signal accumulated in lymphocytes and in a CD11c^hiMHCII^med DC group containing a CD8α^hi subset (non–airway-derived [NAW]-LNDCs). Sorted AW-LNDCs showed long MHCII^bright cytoplasmic processes and intracytoplasmatic FITC⁺ granules. The fraction of FITC⁺ AW-LNDCs peaked after 24 h and had reached baseline by day 7. AW-LNDCs were depleted by 7 d of ganciclovir treatment in thymidine kinase transgenic mice, resulting in a strong reduction of FITC-macromolecule transport into the TLNs. Compared with intrapulmonary DCs, AW-LNDCs had a mature phenotype and upregulated levels of MHCII, B7-2, CD40, and intracellular adhesion molecule (ICAM)-1. In addition, sorted AW-LNDCs from FITC-ovalbumin (OVA)–instilled animals strongly presented OVA to OVA-TCR transgenic T cells. These results validate the unique sentinel role of airway DCs, picking up antigen in the airways and delivering it in an immunogenic form to the T cells in the TLNs.