Nucleoside analogs were investigated for their potential to inhibit cowpox virus (a surrogate for variola and monkeypox viruses) in cell culture and in lethal respiratory infections in mice. Cell culture antiviral activity was determined by plaque reduction assays, with cytotoxicity determined by cell proliferation assays. Selectivity indicies (SI's, 50% cytotoxic concentration divided by 50% virus-inhibitory concentration) were determined for 15 compounds. Three arabinofuranosyl (Ara) nucleosides showed activity in mouse mammary tumor (C127I) cells: guanine (Ara-G), thymine (Ara-T), and adenine (Ara-A) with SI's of 113, 61, and 95, respectively. The 2'-fluoro-Ara nucleosides of 5-F-cytosine (FIAC), 5-methyluracil (FMAC), and 5-iodouracil (FIAU) exhibited SI's of 148, 77, and 29, respectively. Other potent compounds included cidofovir (a positive control) and 3'-O-methyladenosine, with SI values of 164 and 56, respectively. In general, assays performed in African green monkey kidney (Vero) cells produced lower SI's than in C127I cells, except for 5-iodo-2'-deoxyuridine (IDU) which had an SI of > 71 in Vero cells and 3.1 in C127I cells. Intranasal infection of mice with cowpox virus was followed a day later by twice daily intraperitoneal treatment with compounds for 5 days. Ara-A was active at 300 mg/kg/day (40% survival), FAMU at 100 mg/kg/day (70% survival), and cidofovir (given for 1 day only) at 100 mg/kg (80-100% survival). None of the other compounds, including IDU, prevented death nor delayed the time to death. Cidofovir had the best potential for treating orthopoxvirus infections of those tested.