SDS-polyacrylamide gel electrophoretic analysis of plasma membranes prepared from L cells infected with radioiodinated vaccinia virus particles showed that at 2.0 hr postinfection, 125I-labeled virion polypeptides with molecular weights of 58K-60K, 32K-34K, 17K, and 12K-14K were associated with infected cell plasma membranes. By 4 hr postinfection, only the 32- to 34-kDa polypeptide, derived from infecting virus particles, could be detected on infected cell surfaces. A variety of techniques were applied to analyzing purified plasma cell membranes to define the viral antigens expressed on cell surfaces after infection, including (a) surface radioiodination of infected cells; (b) immune or Western blotting; (c) specific immunoprecipitation of viral proteins present in nonionic detergent extracts of membranes purified from [35S]methionine-labeled, virus-infected cells. It was determined that vaccinia virus-specified polypeptides with molecular weights of 78K-82K, 65K, 50K, 42K-45K, 35K-37K, 32K-34K, 30K, 20K, and 17K-18K were expressed by 3 hr postadsorption, on the plasma membranes of infected cells and were accessible to binding by exogenous antiviral antibodies. Viral antigens with molecular weights similar to those expressed on cell surfaces were secreted or shed from infected cells and could be detected in the medium harvested from virus-infected mouse L-cell cultures.