Acute retinal necrosis (ARN) is a rare, potentially blinding retinal disease resulting from ocular infections with herpes simplex virus (HSV) or varicella-zoster virus (VZV). To determine the antigen specificity and functional characteristics of ocular infiltrating T cells in ARN, T cells were isolated and expanded nonspecifically from intraocular fluid (IOF) samples from 2 patients with HSV-1- and 3 with VZV-mediated ARN. HSV-specific T cell reactivity could be detected only in the IOF-derived T cell lines (TCLs) of the 2 patients with HSV-mediated ARN. These TCLs consisted of both HSV type-common and type-specific CD4+ and CD8+ T cell clones (TCCs) with differential T cell receptor usage. Irrespective of their phenotype, the TCCs were cytolytic and secreted interferon-γ, tumor necrosis factor-α, interleukin-4, and interleukin-5. In both patients, the antigen specificity of a substantial number of HSV-1-specific TCCs could be mapped to ∼0.67–0.73 HSV-1 map units. The data presented suggest the contribution of T cells, specific for the triggering virus, to the pathogenesis of ARN.