To visualize the primary antigen-specific T cell response to Ag introduced into the eye, we have used an adoptive transfer system in which a limiting number of OVA peptide (323-339)-specific T cells from a TCR-transgenic mouse were transferred into nonirradiated, syngeneic recipients and then tracked in vivo by staining for FACS analysis or immunohistochemistry with the clonotypic mAb KJ1-26. Following posterior chamber injection of Ag, KJ1-26+ cells accumulated primarily in the draining, submandibular lymph node (LN) within 3 days. Although reduced in number, by day 6 these cells were primarily in the paracortical regions and were able to proliferate and secrete IL-2 in response to Ag stimulation. In contrast, following i.v. injection of Ag, the KJ1-26+ cells accumulated in the paracortical regions of the LN to a comparable degree, but did not proliferate or secrete IL-2. The day 3 accumulation of KJ1-26+ cells in the submandibular LN was inhibited if the eye was removed within 5 h after injection of Ag. In the spleen, foci of KJ1-26+ cells were observed in the periarteriolar lymphoid sheaths at day 3; these were not observed to the same degree following other forms of immunization. These results demonstrate that the submandibular LN is the primary site for early clonal expansion of antigen-specific T cells following intraocular Ag administration and that these cells show changes consistent with immunity rather than tolerance.