Purpose: To investigated whether topical tumor necrosis factor-alpha (TNF-α)-antisense-oligonucleotides (ASON) treatment might affect the course of contralateral HSV-retinitis. Methods: In vivo uptake was determined after subconjunctival (s.c.) injection of FITC-labeled TNF-α-ASON. BALB/c mice were injected in the AC of the right eyes with HSV-1 (KOS); the left eyes were injected s.c. 3 times with TNF-α-ASON; sequence-unspecific control (C) ON or buffer. The clinical course of HSV-R, ocular inflammatory cell-infiltration, uptake of [3H]thymidine from rLN cells and viral replication in the eyes were analyzed. Results: In vivo, FITC-TNF-α-ASON was found in the choroid and retina up to 7 days after s.c. injection, but also in the rLN. After s.c. TNF-α-ASON injection, expression of TNF-α was reduced in the ipsilateral eyes and rNL. TNF-α-ASON injection reduced the incidence and severity of early chorioretinitis and of the inflammatory cell infiltration and CD-11b cells in the choroid and retina of the treated eye. On day 10 PI, the virus titers, inflammatory cell infiltration and HSV-R had increased in the TNF-α-ASON treated eyes, while the HSV-1 specific [3H]thymidine uptake from rLN cells was decreased and DTH response did not differ between the PBS, CON and TNF-α-ASON groups. Conclusions: Topical TNF-α-ASON-injection reduced early ocular inflammatory cell infiltration, but delayed the virus clearance and increased the severity of HSV-retinitis in the treated eyes. The antisense technique is a useful experimental approach for the topical application of TNF-α in experimental HSV-1 retinitis.