We have used fluorescent latex beads, with or without covalently conjugated OVA, to facilitate study of Ag trafficking in the mouse lung and draining peribronchial lymph node (LN). At 6 h, and up to 48 h after intranasal administration, beads were observed as intracellular clusters in the tissue parenchyma. Flow cytometry of bead-positive (bead+) cells from the bronchoalveolar lavage demonstrated that a majority of these cells are CD11c+, F4/80+, and CD11b−. Furthermore, fluorescent microscopy confirmed that a major subset of bead+ cells in the lung tissue was also CD11c+. In the draining peribronchial LNs, small numbers of beads were present in the subcapsular sinus as early as 6 h after inhalation. By 12 h and beyond, bead+ cells had localized exclusively to the LN T zone. OVA-conjugated latex beads, in addition to stimulating brisk proliferation of naive, OVA-specific DO11. 10 transgenic T cells in vitro, could also recruit OVA-specific T cells in vivo. In some cases, bead+ APCs and CD4+ Th1 cells were found adjacently localized in the lung tissue 6 h after airway challenge. Thus, interactions of bead+ APCs with Ag-specific CD4+ T cells occurred earlier in the peripheral airways than these same interactions occurred in the draining peribronchial LN. Lastly, after adoptive transfer, in vitro differentiated Th1 cells accumulated at peripheral sites in the lung tissue and airways before Ag challenge and therefore were ideally positioned to influence subsequent immune reactions of the airway.