Currently there is no clinically effective antiviral agent for the prevention or treatment of ocular adenoviral infections. Using a paired-eye, masked design, we tested the antiviral efficacy of topical 0.1% (S)-1-(3-hydroxy-2-phosphonylmethoxypropyl)cytosine in the New Zealand rabbit ocular model after topical and intrastromal inoculation with 100 microliters (4 x 10(5) plaque-forming units per eye) of adenovirus type 5 McEwen, a clinical isolate. Prevention studies involved pretreatment (six times a day) 1 day before inoculation and continuing for 4 additional days. Compared with the control eyes, the pretreated eyes showed a significant reduction in the peak viral eye titers on days 3, 4, 5, and 7 after treatment (p < 0.03-0.005), and a reduction in the duration of viral sheeding (p < 0.02). Rebound increase in adenoviral titers was detected in five of 20 eyes (25%) after cessation of treatment, suggesting a therapeutic effect and a need for further studies to optimize the treatment regimen.