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Herpes stromal keratitis (HSK) results from the reactivation of herpes simplex virus type-1 (HSV-1) in the cornea. The subsequent corneal inflammation and neovascularization may lead to scarring and visual loss. The cellular and molecular mechanisms underlying HSK remain unknown. The presence of stromal HSV-1 viral proteins or antigens in the HSK cornea remains a subject of debate. It was recently reported that HSV-1 ICP0 rapidly diffuses out of infected rabbit corneas. To investigate further the presence of HSV-1 ICP0 in the infected cornea, particularly in the corneal stroma, ex vivo confocal microscopy was used to scan rabbit corneas infected with the virus ICP0–EYFP, an HSV-1 derivative (strain 17+) that expresses ICP0 fused to the enhanced yellow fluorescent protein (EYFP). These results demonstrate that ICP0 is expressed in the corneal epithelium and stromal cells (keratocytes) of infected rabbit corneas throughout acute infection. Furthermore, expression of ICP0–EYFP appears localized to punctate, granular deposits within stromal keratocytes, showing both a cytoplasmic and perinuclear localization. These findings provide new data demonstrating that anterior corneal keratocytes become infected and express ICP0 during acute HSV-1 infection.