The combination of ST-246 and hexadecyloxypropyl-cidofovir or CMX001 was evaluated for synergistic activity in vitro against vaccinia virus and cowpox virus (CV) and in vivo against CV. In cell culture the combination was highly synergistic against both viruses, and the results suggested that combined treatment with these agents might offer superior efficacy in vivo. For animal models, ST-246 was administered orally with or without CMX001 to mice lethally infected with CV. Treatments began 1, 3, or 6 days postinfection using lower dosages than previously used for single-drug treatment. ST-246 was given at 10, 3, or 1 mg/kg of body weight with or without CMX001 at 3, 1, or 0.3 mg/kg to evaluate potential synergistic interactions. Treatment beginning 6 days post-viral inoculation with ST-246 alone only increased the mean day to death at 10 or 3 mg/kg but had no effect on survival. CMX001 alone also had no effect on survival. When the combination of the two drugs was begun 6 days after viral infection using various dosages of the two, a synergistic reduction in mortality was observed. No evidence of increased toxicity was noted with the combination either in vitro or in vivo. These results indicate that combinations of ST-246 and CMX001 are synergistic both in vitro and in vivo and suggest that combination therapy using ST-246 and CMX001 for treatment of orthopoxvirus disease in humans or animals may provide an additional benefit over the use of the two drugs by themselves.