Poxviruses that are serologically related to vaccinia virus produce in addition to common antigens three precipitable specific antigenic substances: vc, va, and mo. These antigens were recognized by spur formation in immunodiffusion. Their specificity was confirmed by antibody production and absorption tests. By identification of the antigens with specific antisera, these poxviruses were classified in three groups: variola (vc−, va+, mo−), monkeypox (vc−, va−, mo+), and vaccinia (vc+, va+, mo−), Cowpox tissue extracts did not react with any of the specific sera. Three variola-like isolates from healthy monkeys could be classified serologically as variola. Extracts of tissues infected with rabbitpox, buffalopox, and camelpox reacted like vaccinia. White mutants from two monkeypox isolates produced the same specific antigen as their parent strains.