Avian poxvirus was isolated from nodules on the heads and conjunctiva of two 3-to-4-wk-old ostrich chicks. The ostriches from which poxvirus was isolated had been placed on premises where turkeys that had shown evidence of poxvirus infection had been raised earlier. Microscopically, the nodules from the ostriches were composed of proliferating and hypertrophic epithelial cells that formed large fronds. Most of the hypertrophic epithelial cells contained large eosinophilic intracytoplasmic inclusion bodies characteristic of poxvirus. Characterization of the avian poxvirus isolated from the cutaneous lesions in ostriches was based on western blotting of virus antigen, restriction fragment length polymorphism of genomic DNA, pathogenesis, and cross-protection studies in chickens. Antigenic and genetic studies did not reveal any significant difference between the poxvirus isolated from ostriches (PVO) and fowl poxvirus (FPV). Further, susceptible chickens immunized with the PVO were protected when challenged with a virulent strain of FPV. Thus, the poxvirus isolated from ostriches had similar antigenic, genetic, and biological properties to FPV.