The acyclovir-induced herpes simplex virus Type 1 (HSV-1) strain, R9C2, a double mutant in thymidine kinase (TK) and DNA polymerase (DNA pol), and its parental strain SC16 were compared for their effects on ocular pathology and systemic immunity after unilateral inoculation into the anterior chamber (AC) of BALB/c mouse eyes. Although AC-injected R9C2produced no retinal necrosis (0/18 eyes), this mutant induced active suppression (33–87%) of anti–HSV delayed type hypersensitivity similar to that induced by another HSV strain, KOS. AC-injected parental strain, SC16, caused fatal disease within 7–10 days, and induced bilateral retinal necrosis and suppression of DTH in 100% of the mice. Preimmunization with R9C2protected mice in a dose-dependent fashion from the pathologic and lethal effects of AC-injected parental virus. These data suggest that the immunogenicity of the TK and DNA pol double mutant remains intact despite the decreased ocular and systemic pathogenicity observed after intracameral inoculation.