Herpetic stromal keratitis (HSK) is a corneal disease initiated by a herpes simplex virus (HSV) infection with a postulated T cell-mediated immunopathology. To study the antigen specificity of cornea-infiltrating T cells in HSK patients, T cells were isolated and expanded by mitogenic stimulation from corneas of 2 patients with HSV-1-mediated HSK. A substantial number of the T cell clones (TCCs) obtained from these T cell lines were HSV-specific. All HSV-specific TCCs were of the CD3⁺CD4⁺CD8⁻ phenotype. These TCCs responded to autologous HSV-infected corneal keratocytes, which expressed HLA class II molecules following incubation with interferon-γ. Upon HSV-specific stimulation, all TCCs secreted interleukin-4, interleukin-5, and interferon-γ. The data presented suggest that HSV-specific CD4⁺ T cells play a role in the immunopathogenesis of HSK in humans and that corneal keratocytes may act as antigen-presenting cells in this local T cell response.