Experimental autoimmune uveitis (EAU) is an inflammatory disease of the immune privileged inner eye, mediated by CD4⁺ Th1 cells specific for retinal autoantigens. To elucidate the fate of the T cells in the eye we adoptively transferred green fluorescent protein-positive (GFP⁺) T cells with specificity for R14, a peptide from interphotoreceptor retinoid-binding protein (IRBP) or OVA as foreign control antigen to naive Lewis rats. We also used the model of immunogenic uveitis, an inflammatory eye disease induced by intraocular injection of soluble OVA 1 day post transfer of OVA-specific GFP⁺ cells. We investigated the timing of ocular T cell infiltration and their immunological activation state by intravital fluorescence microscopy (IVFM) of the iris until onset of intraocular inflammation. Within 30 min of injection, GFP⁺ cells invaded the iris tissue, irrespective of their antigen specificity, whereas intraocular inflammation was only observed 3 days later, if cells recognized their respective antigen (R14-specific cells in EAU, OVA-specific cells in immunogenic uveitis). Using FACS analysis we found that activation markers were upregulated only on cells from uveitic eyes, but not from other sources, suggesting that intraocularly presented specific antigen is a prerequisite for T cell reactivation and subsequent recruitment of inflammatory cells.