Studies of experimental infection of prairie dogs (Cynomys ludovicianus) with monkeypox virus are described. After intraperitoneal infection, all of the animals died within 11 days. Virus was cultured from their blood and oropharynx several days before death; at necropsy, most of the organs tested contained monkeypox virus. Marked hepatic and splenic necrosis were observed, along with mild inflammatory changes in the lungs. After intranasal (IN) infection, the primary pathologic changes were in the lungs and pleural cavity. Some of the IN infected animals (40%) survived, and monkeypox virus could be cultured from their nasal discharge and oropharynx for <22 days. Ulcerative lesions also developed on the lips, tongue, and buccal mucosa of the surviving animals. Our findings support an earlier report, which suggested that infected prairie dogs can transmit monkeypox virus by respiratory and mucocutaneous contact with susceptible animals and persons.