Antigenic materials prepared from vaccinia infected tissues or cell cultures have been studied by diffusion-in-gel methods. Using an immune serum produced by hyperimmunization of a rabbit and employed as a reference serum and a material prepared from vaccinia infected rabbit skin, employed as a reference antigen, 8 separate precipitinogenic factors were revealed. The immunological relation between these factors and those produced in vaccinia or variola infected CAM, vaccinia infected HeLa cells and vaccinia infected bovine embryonic skin-muscle tissue was analyzed.