A novel carbocyclic thymidine analog, N-methanocarbathymidine [(N)-MCT], was evaluated for inhibition of orthopoxvirus infections. Efficacy in vitro was assessed by plaque reduction assays against wild-type and cidofovir-resistant strains of cowpox and vaccinia viruses in nine different cell lines. Minimal differences were seen in antiviral activity against wild-type and cidofovir-resistant viruses. (N)-MCT's efficacy was affected by the cell line used for assay, with 50% poxvirus-inhibitory concentrations in cells as follows: mouse = 0.6–2.2 μM, rabbit = 52–90 μM, monkey = 87 to >1000 μM, and human = 39–220 μM. Limited studies performed with carbocyclic thymidine indicated a similar cell line dependency for antiviral activity. (N)-MCT did not inhibit actively dividing uninfected cells at 1000 μM. The potency of (N)-MCT against an S-variant thymidine kinase-deficient vaccinia virus was similar to that seen against S-variant and wild-type viruses in mouse, monkey, and human cells, implicating a cellular enzyme in the phosphorylation of the compound. Mice were intranasally infected with cowpox and vaccinia viruses followed 24 h later by intraperitoneal treatment with (N)-MCT (twice a day for 7 days) or cidofovir (once a day for 2 days). (N)-MCT treatment at 100 and 30 mg/kg/day resulted in 90 and 20% survival from cowpox virus infection, respectively, compared to 0% survival in the placebo group. Statistically significant reductions in lung virus titers on day 5 occurred in 10, 30, and 100 mg/kg/day treated mice. These same doses were also active against a lethal vaccinia virus (WR strain) challenge, and protection was seen down to 10 mg/kg/day against a lethal vaccinia virus (IHD strain) infection. Cidofovir (100 mg/kg/day) protected animals from death in all three infections.